Systems and devices for sequence by synthesis analysis

ABSTRACT

The present invention comprises systems and devices for sequencing of nucleic acid, such as short DNA sequences from clonally amplified single-molecule arrays.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to and is a national stage entryof International Patent Application No. PCT/US2007/007991, which claimspriority to U.S. Provisional Patent Application Nos. 60/788,248, filedMar. 31, 2006, and 60/795,368 filed Apr. 26, 2006, all of which areherein incorporated by reference in their entirety for all purposes.

FIELD OF THE INVENTION

The current invention relates to the field of nucleic acid sequencing.More specifically, the present invention provides systems and devicesfor sequence analysis of nucleic acids such as short DNA sequences fromclonally amplified single-molecule arrays.

BACKGROUND OF THE INVENTION

Numerous recent advances in the study of biology have benefited fromimproved methods of analysis and sequencing of nucleic acids. Forexample, the Human Genome Project has determined the entire sequence ofthe human genome which is hoped to lead to further discoveries in fieldsranging from treatment of disease to advances in basic science. Whilethe “human genome” has been sequenced there are still vast amounts ofgenomic material to analyze, e.g., genetic variation between differentindividuals, tissues, additional species, etc.

Devices for DNA sequencing based on separation of fragments of differinglength were first developed in the 1980s, and have been commerciallyavailable for a number of years. However, such technology involvesrunning individual samples through capillary columns filled withpolyacrylamide gels and is thus limited in throughput due to the timetaken to run each sample. A number of new DNA sequencing technologieshave recently been reported that are based on the massively parallelanalysis of unamplified (WO00006770: Proceedings of the National Academyof Sciences U.S.A. 100, 3960-3964 (2003)) or amplified single molecules,either in the form of planar arrays (WO9844151) or on beads (WO04069849:Nature, 437, 376-380 (2005): Science, 309, 5741, 1728-1732 (2005); NatBiotechnol. 6, 630-6344 (2000)).

The methodology used to analyze the sequence of the nucleic acids insuch new sequencing techniques is often based on the detection offluorescent nucleotides or oligonucleotides. The detectioninstrumentation used to read the fluorescence signals on such arrays isusually based on either epifluorescence or total internal reflectionmicroscopy, for example as described in WO9641011, WO00006770 orWO02072892. Whilst total internal reflection microscopy has been used toimage both single and amplified molecules of DNA on surfaces, a robust,reliable, four color DNA sequencing platform (e.g., comprising heatingsystems, fluidic controls, uniform illumination, control of the opticalbeam shape, an autofocus system, and hill software control of allcomponents) is described herein for the first time.

There is a continuing need for better, more robust, and more economicaldevices and systems for fast reliable sequencing of nucleic acids. Thecurrent invention provides these and other benefits which will beapparent upon examination of the current specification, claims, andfigures.

SUMMARY OF THE INVENTION

In various aspects herein, the invention comprises systems and devicesfor sequencing one or more polynucleotide. The systems can be used toimage planar substrates, wherein the substrates can comprise unamplifiedsingle molecules, amplified single molecules, one or more collections ofarrayed beads, or various combinations thereof. When used forsequencing, the systems can optionally comprise a planar solid substratehaving one or more polynucleotides displayed thereon, e.g. eitherdirectly attached, or attached to beads that are optionally arrayed onthe substrate; a fluid direction system that controllably moves variousreagents (e.g., buffers, enzymes, fluorescently labeled nucleotides oroligonucleotides, etc.) into contact with the polynucleotides; atemperature control system that regulates the temperature of thesubstrate and/or of the reagents; an optical system for obtaining totalinternal reflection illumination of the substrate with a uniform beamfootprint (where the shape of the footprint is optionally controlled), alight source (e.g., one comprising one or more lasers) for exciting thefluorescent moiet(ies); a detector component (e.g., a CCD camera andobjective lenses, etc.) that is proximal to the substrate and whichcaptures and detects fluorescence from the excited moiet(ies); acomputer, connected to the detector, which has instruction sets forcontrolling the various components of the system, acquiring fluorescencedata from the detector and optionally for determining sequence of thepolynucleotide from the fluorescence data.

In some such embodiments, the substrates can be moved away from thedetector in order to interact with the temperature control system, thus,regulating the temperature of the substrate (e.g., to allow polymerasereactions to proceed, etc.). In such embodiments, the system cancomprise a scanning stage or moving platform that is optionally computercontrolled. The heating device can be a computer controlled Peltierdevice or other heating/cooling component that moves in relation to thescanning stage, or the stage can optionally move to ensure that thePeltier is in contact with the substrate.

In the various embodiments herein, the substrate can comprise aflowcell. Flowcells can have one or more fluidic channel in which thepolynucleotide is displayed (e.g., wherein the polynucleotides aredirectly attached to the flowcell or wherein the polynucleotides areattached to one or more beads arrayed upon the flowcell) and can becomprised of glass, silicon, plastic, or various combinations thereof.

In typical embodiments, the reagents include components to synthesize asecond sequence complementary to the one or more polynucleotides. Thesynthesis can be performed using labeled nucleotides, which can be addedindividually or as a mixture of nucleotides, or as labeledoligonucleotides. In the case of labeled oligonucleotides, the identityof one or more bases complementary to the labeled oligonucleotide can bedetermined. The labeled nucleotides can take the form of fluorescentlylabeled triphosphates, which can contain a blocking moiety to controlthe addition and ensure a single nucleotide is added to eachpolynucleotide. The fluorophore can be attached to the blocking moiety,which can be located at the 3′ position of the sugar, or can be attachedthrough the nucleotide base through a linker that can optionally becleaved using the same conditions as removal of the blocking moiety. Thelinker and blocking moiety may be cleaved using the same reagents.

In various embodiments herein, the Total Internal Reflection (TIRF)system can comprise, e.g., a lamp or a laser. The system can comprisemore than one excitation lasers that can be coupled through a fiberopticdevice. Such lasers can illuminate at least part of the same area.(i.e., overlap). The TIRF lasers herein also optionally comprise ashaking, vibrating, waveplate modulated, or piezo-electric actuatorsqueezed fiber mode scrambler to make the optical intensitysubstantially uniform over an entire illumination footprint of thelaser. A number of mechanisms for controlling the illumination intensityand uniformity are described herein. The shape of the fiber also can beused to control the shape of the illumination footprint.

The detector component in the various embodiments herein can compriseone or more objective lenses, additional tube lenses, an autofocussystem that adjusts either the stage position and/or the position of theobjective lens(es) to ensure the substrate remains in focus, opticalfilter(s) appropriate to transmit the emission wavelength of thefluorophores and block the light from the excitation source, and asystem for recording the fluorescence emission from the fluorophores,for example a charge coupled device (CCD) or similar camera.

These and other features of the invention will become more fullyapparent when the following detailed description is read in conjunctionwith the accompanying figures and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, displays a generalized overview of the major components of anexemplary system of the invention.

FIG. 2, displays a photograph of an exemplary system of the inventionshown without an enclosing chassis or covering.

FIG. 3, displays a photograph of an exemplary flowcell, lens objective,and fiber optic laser arrangement within a system of the invention.

FIG. 4, Panels A-D show exemplary configurations of flowcells.

FIG. 5, Panels A and B, show one method of forming a flowcell of thesystem (Panel A) and a transmission spectra of Foturan glass (Panel B).

FIG. 6, Panels A-E show an exemplary possible etching method toconstruct flowcells herein.

FIG. 7, Panels A-C present exemplary schematic diagrams of possiblefluid flow components/arrangements of the system in push (Panel A) orpull (Panels B and C) configurations.

FIG. 8, shows an exemplary heating/cooling component of the system inisolation from other aspects of the invention.

FIG. 9, panels A-D present schematic diagrams of possible flowed andflowcell holder configurations of the invention.

FIG. 10, Panels A and B present photographs of an exemplary embodimentof the invention showing movement of the heating/cooling component andthe flowcell holder (Panel A) and a schematic of the heating/coolingcomponents in relation to other components of an exemplary system (PanelB).

FIG. 11, Panels A and B show schematics displaying an exemplaryframework holding the optics, fiber optic laser mount, heating/cooling,and flowcell holder components (A); and an exemplary flowcell levelingadjustment configuration (B).

FIG. 12, presents a picture of an exemplary embodiment of the systemshowing framework and housing of the system.

FIGS. 13-16, present various optional configurations of cameras, lightsources, and other components in the systems herein.

FIGS. 17-19, show various schematics for beam shape and dimensions forTIRF lasers in various embodiments of the systems herein.

FIG. 20, displays an optional embodiment of a TIRF prism for use withthe systems and devices herein.

FIG. 21, illustrates creation of a square laser beam by polishing theend of a multimode fiber output.

FIG. 22, Panels A and B, illustrate an exemplary filters and filterwheel configuration optionally within various embodiments herein (A), aswell as the spectrum of those filters in relation to four exemplaryfluorophores excited at the laser wavelengths (B).

FIG. 23, illustrates an exemplary nominal 1G design, 30× K4 System Raytrace of the optic components of a system of the invention.

FIG. 24, shows the 30× K4 imaging performance of an exemplary system ofthe invention.

FIG. 25, presents a schematic diagram of an autofocusing feature of anexemplary system herein.

FIGS. 26-27, display photographs of focused and unfocused measurementsmade by various embodiments of the systems/devices herein.

FIG. 28, presents a diagram of an auto focus laser beam.

FIG. 29, shows a graph of the number of detected nucleic acid clustersas a function of total cluster number and minimum cluster area asdetected by an embodiment of the invention.

FIGS. 30-32, display outlines of nucleic acid clusters and theirsequencing with the systems/devices of the invention.

FIG. 33, Panels A-D show the effects of three different forms ofphysical deformation to a circular optical fiber on the beam emergingfrom the fiber. Vibrating or squeezing the fiber makes the lightemerging from the fiber uniform over the integration time of the image.

FIG. 34, Panels A-D show the effects of three different forms ofphysical deformation to a rectangular optical fiber on the beam emergingfrom the fiber. Vibrating or squeezing the fiber makes the lightemerging from the fiber uniform over the integration time of the image.

FIG. 35, Panels A-L display the effects of various mode scramblingschemes on emergent light from a number of different optical fibers.

FIG. 36 shows one possible arrangement for a dual camera systemembodiment of the invention.

FIG. 37 shows an exemplary embodiment of the invention containing 2cameras for simultaneous recording of 2 colors on the same image.

FIG. 38, shows a schematic of a λ/2 waveplate.

FIG. 39, shows a schematic of a λ/2 modified waveplate comprising anumber of differently orientated sections.

FIG. 40, shows an outline of a mode waveplate modulated mixing system ofan embodiment of the invention.

FIG. 41, Panels A-D show photographs of an illuminated footprint areafrom a multimode optical fiber and results from mixing of optical modesthrough use of waveplates.

FIG. 42, Panels A and B display the substantial uniformity of a laserfootprint area from multimode mixing through use of waveplates.

FIG. 43 shows a dual flowcell holder embodiment of the invention suchthat chemistry operations can be performed in parallel in order tomaximize the scanning time of the instrument.

FIG. 44, Panels A-F show exemplary embodiments of bottom flow flowcells,prisms, and side/top TIRF illumination

FIG. 45 shows an exemplary temperature regulation component beneath aflowcell and prism.

FIG. 46 shows an exemplary fluidic valve and exemplary manifolds (e.g.,for use with bottom flow flowcells.

FIG. 47 shows an exemplary fluidic valve of the invention.

FIG. 48, Panels A and B show one possible dual flowcell configuration ofthe invention.

FIG. 49, Panels A-F show various exemplary bottom temperature regulationconfigurations capable of use with bottom flow flowcells of theinvention.

DETAILED DESCRIPTION

The present invention comprises systems and devices to analyze a largenumber of different nucleic acid sequences from, e.g., clonallyamplified single-molecule DNA arrays in flowcells, or from an array ofimmobilized beads. The systems herein are optionally useful in, e.g.,sequencing for comparative genomics (such as for genotyping, SNPdiscovery, BAC-end sequencing, chromosome breakpoint mapping, and wholegenome sequence assembly), tracking gene expression, micro RNA sequenceanalysis, epigenomics (e.g., with methylation mapping DNAselhypersensitive site mapping or chromatin immunoprecipitation), andaptamer and phage display library characterization. Of course, those ofskill in the art will readily appreciate that the current invention isalso amenable to use for myriad other sequencing applications. Thesystems herein comprise various combinations of optical, mechanical,fluidic, thermal, electrical, and computing devices/aspects which aredescribed more fully below. Also, even though in certain embodiments theinvention is directed towards particular configurations and/orcombinations of such aspects, those of skill in the art will appreciatethat not all embodiments necessarily comprise all aspects or particularconfigurations (unless specifically stated to do so).

In brief, the general aspects of the invention are outlined in FIG. 1which shows an exemplary TIRF imaging configuration of a backlightdesign embodiment. As can be seen in FIG. 1, fluid delivery module ordevice 100 directs the flow of reagents (e.g., fluorescent nucleotides,buffers, enzymes, cleavage reagents, etc.) to (and through) flowcell 110and waste valve 120. In particular embodiments, the flowcell comprisesclusters of nucleic acid sequences (e.g., of about 200-1000 bases inlength) to be sequenced which are optionally attached to the substrateof the flowcell, as well as optionally other components. The flowcellcan also comprise an array of beads, where each bead optionally containsmultiple copies of a single sequence. The preparation of such beads canbe performed according to a variety of techniques, for example asdescribed in U.S. Pat. No. 6,172,218 or WO04069849 (Bead emulsionnucleic acid amplification).

The system also comprises temperature station actuator 130 andheater/cooler 135, which can optionally regulate the temperature ofconditions of the fluids within the flowcell. As explained below,various embodiments can comprise different configurations of theheating/cooling components. The flowcell is monitored, and sequencing istracked, by camera system 140 (e.g., a CCD camera) which can interactwith various filters within filter switching assembly 145, lensobjective 142, and focusing laser/focusing laser assembly 150. Laserdevice 160 (e.g., an excitation laser within an assembly optionallycomprising multiple lasers) acts to illuminate fluorescent sequencingreactions within the flowcell via laser illumination through fiber optic161 (which can optionally comprise one or more re-imaging lenses, afiber optic mounting, etc. Low watt lamp 165, mirror 180 and reversedichroic 185 are also presented in the embodiment shown. See below.Additionally, mounting stage 170, allows for proper alignment andmovement of the flowcell, temperature actuator, camera, etc. in relationto the various components of the invention. Focus (z-axis) component 175can also aid in manipulation and positioning of various components(e.g., a lens objective). Such components are optionally organized upona framework and/or enclosed within a housing structure. It will beappreciated that the illustrations herein are of exemplary embodimentsand are not necessarily to be taken as limiting. Thus, for example,different embodiments can comprise different placement of componentsrelative to one another (e.g., embodiment A comprises a heater/cooler asin FIG. 1, while embodiment B comprises a heater/cooler componentbeneath its flowcell, etc.).

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that the invention herein is not limited to use withparticular nucleic acids or biological systems, which can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. As used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a flowcell” optionally includes a combination of two ormore flowcells, and the like.

As used herein, the terms “polynucleotide” or “nucleic acids” refer todeoxyribonucleic acid (DNA), but where appropriate the skilled artisanwill recognize that the systems and devices herein can also be utilizedwith ribonucleic acid (RNA). The terms should be understood to include,as equivalents, analogs of either DNA or RNA made from nucleotideanalogs. The terms as used herein also encompasses cDNA, that iscomplementary, or copy, DNA produced from an RNA template, for exampleby the action of reverse transcriptase.

The single stranded polynucleotide molecules sequenced by the systemsand devices herein can have originated in single-stranded form, as DNAor RNA or have originated in double-stranded DNA (dsDNA) form (e.g.genomic DNA fragments, PCR and amplification products and the like).Thus a single stranded polynucleotide may be the sense or antisensestrand of a polynucleotide duplex. Methods of preparation of singlestranded polynucleotide molecules suitable for use in the method of theinvention using standard techniques are well known in the art. Theprecise sequence of the primary polynucleotide molecules is generallynot material to the invention, and may be known or unknown. The singlestranded polynucleotide molecules can represent genomic DNA molecules(e.g., human genomic DNA) including both intron and exon sequences(coding sequence), as well as non-coding regulatory sequences such aspromoter and enhancer sequences.

In certain embodiments, the nucleic acid to be sequenced through use ofthe current invention is immobilized upon a substrate (e.g., a substratewithin a flowcell or one or more beads upon a substrate such as aflowcell, etc.). The term “immobilized” as used herein is intended toencompass direct or indirect, covalent or non-covalent attachment,unless indicated otherwise, either explicitly or by context. In certainembodiments of the invention covalent attachment may be preferred, butgenerally all that is required is that the molecules (e.g. nucleicacids) remain immobilized or attached to the support under conditions inwhich it is intended to use the support, for example in applicationsrequiring nucleic acid sequencing.

The term “solid support” (or “substrate” in certain usages) as usedherein refers to any inert substrate or matrix to which nucleic acidscan be attached, such as for example glass surfaces, plastic surfaces,latex, dextran, polystyrene surfaces, polypropylene surfaces,polyacrylamide gels, gold surfaces, and silicon wafers. In manyembodiments, the solid support is a glass surface (e.g., the planarsurface of a flowcell channel). In certain embodiments the solid supportmay comprise an inert substrate or matrix which has been“functionalized,” for example by the application of a layer or coatingof an intermediate material comprising reactive groups which permitcovalent attachment to molecules such as polynucleotides. By way ofnon-limiting example such supports can include polyacrylamide hydrogelssupported on an inert substrate such as glass. In such embodiments themolecules (polynucleotides) can be directly covalently attached to theintermediate material (e.g. the hydrogel) but the intermediate materialcan itself be non-covalently attached to the substrate or matrix (e.g.the glass substrate). Covalent attachment to a solid support is to beinterpreted accordingly as encompassing this type of arrangement.

System Overview

As indicated above, the present invention comprises novel systems anddevices for sequencing nucleic acids. As will be apparent to those ofskill in the art, references herein to a particular nucleic acidsequence may, depending on the context, also refer to nucleic acidmolecules which comprise such nucleic acid sequence. Sequencing of atarget fragment means that a read of the chronological order of bases isestablished. The bases that are read do not need to be contiguous,although this is preferred, nor does every base on the entire fragmenthave to be sequenced during the sequencing. Sequencing can be carriedout using any suitable sequencing technique, wherein nucleotides oroligonucleotides are added successively to a free 3′ hydroxyl group,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. The nature of the nucleotide added is preferably determinedafter each nucleotide addition. Sequencing techniques using sequencingby ligation, wherein not every contiguous base is sequenced, andtechniques such as massively parallel signature sequencing (MPSS) wherebases are removed from, rather than added to, the strands on the surfaceare also amenable to use with the systems and devices of the invention.

In certain embodiments, the current invention utilizessequencing-by-synthesis (SBS). In SBS, four fluorescently labeledmodified nucleotides are used to sequence dense clusters of amplifiedDNA (possibly millions of clusters) present on the surface of asubstrate (e.g., a flowcell). The inventors and coworkers have describedvarious additional aspects regarding SBS procedures and methods whichcan be utilized with the systems and devices herein. See, e.g.,WO04018497, WO04018493 and U.S. Pat. No. 7,057,026 (nucleotides),WO05024010 and WO06120433 (polymerases), WO05065814 (surface attachmenttechniques), and WO 9844151, WO06064199 and WO07010251, the contents ofeach of which are incorporated herein by reference in their entirety.

In particular uses of the systems/devices herein the flowcellscontaining the nucleic acid samples for sequencing are placed within theappropriate flowcell holder of the present invention (variousembodiments of which are described herein). The samples for sequencingcan take the form of single molecules, amplified single molecules in theform of clusters, or beads comprising molecules of nucleic acid. Thenucleic acids are prepared such that they comprise an oligonucleotideprimer adjacent to an unknown target sequence. To initiate the first SBSsequencing cycle, one or more differently labeled nucleotides, and DNApolymerase, etc., are flowed into/through the flowcell by the fluid flowsubsystem (various embodiments of which are described herein). Either asingle nucleotide can be added at a time, or the nucleotides used in thesequencing procedure can be specially designed to possess a reversibletermination property, thus allowing each cycle of the sequencingreaction to occur simultaneously in the presence of all four labelednucleotides (A, C, T, G). Where the four nucleotides are mixed together,the polymerase is able to select the correct base to incorporate andeach sequence is extended by a single base. In such methods of using thesystems of the invention, the natural competition between all fouralternatives leads to higher accuracy than wherein only one nucleotideis present in the reaction mixture (where most of the sequences aretherefore not exposed to the correct nucleotide). Sequences where aparticular base is repeated one after another (e.g., homopolymers) areaddressed like any other sequence and with high accuracy.

The fluid flow subsystem also flows the appropriate reagents to removethe blocked 3′ terminus (if appropriate) and the fluorophore from eachincorporated base. The substrate can be exposed either to a second roundof the four blocked nucleotides, or optionally to a second round with adifferent individual nucleotide. Such cycles are then repeated and thesequence of each cluster is read over the multiple chemistry cycles. Thecomputer aspect of the current invention can optionally align thesequence data gathered from each single molecule, cluster or bead todetermine the sequence of longer polymers, etc. Alternatively, the imageprocessing and alignment can be performed on a separate computer.

The heating/cooling components of the system regulate the reactionconditions within the flowcell channels and reagent storageareas/containers (and optionally the camera, optics, and/or othercomponents), while the fluid flow components allow the substrate surfaceto be exposed to suitable reagents for incorporation (e.g., theappropriate fluorescently labeled nucleotides to be incorporated) whileunincorporated reagents are rinsed away. An optional movable stage uponwhich the flowcell is placed allows the flowcell to be brought intoproper orientation for laser (or other light) excitation of thesubstrate and optionally moved in relation to a lens objective to allowreading of different areas of the substrate. Additionally, othercomponents of the system are also optionally movable/adjustable (e.g.,the camera, the lens objective, the heater/cooler, etc.). During laserexcitation, the image/location of emitted fluorescence from the nucleicacids on the substrate is captured by the camera component, thereby,recording the identity, in the computer component, of the first base foreach single molecule, cluster or bead.

FIG. 2 displays a photograph of an exemplary arrangement of a system ofthe invention. As can be seen, the system can be divided into severalbasic groupings, e.g., area 200 comprising fluidics and reagent storage(including pumps and motors or the like for producing and regulatingfluid flow, heaters/coolers for proper reagent temperatures, etc.), area210 comprising flowcell and detection (including one or more cameras orsimilar devices, one or more lasers or other light sources, one or moreappropriate optical filters and lenses, a temperature control actuator,e.g., with Peltier heating/cooling for control of the temperatureconditions of the flowcell, a movable staging platform and motorscontrolling such to correctly position the various devices/componentswithin the system), and area 220 comprising a computer module (includingmemory and a user interface such as a display panel and keyboard, etc.).

FIG. 3 shows a photograph of a flowcell (flowcell 300) placed within anexemplary system. A laser coupled through optical fiber 320 ispositioned to illuminate the flowcell (which contains the nucleic acidsamples to be sequenced) while an objective lens component (component310) captures and monitors the various fluorescent emissions once thefluorophores are illuminated by a laser or other light. Also as can beseen in FIG. 3, reagents are flowed through the flowcell through one ormore tubes (tube 330) which connect to the appropriate reagent storage,etc. The flowcell in FIG. 3 is placed within flowcell holder 340 (whichis, in turn, placed upon movable staging area 350). The flowcell holderkeeps the flowcell secure in the proper position in relation to thelaser, the prism (which directs laser illumination onto the imagingsurface), and the camera system, while the sequencing occurs. Otherflowcells and flowcell configurations are set forth below.

The various embodiments of the current invention present several novelfeatures (again, it will be appreciated that not all features arenecessarily present in all embodiments unless specifically stated to beso). For example, the systems herein can use two excitation laserscoupled through a fiberoptic device to ensure that they illuminate thesame area (i.e. that the illuminated areas, or footprints, of the lasersoverlap). Additionally, the current invention can contain a shaking,squeezed, or waveplate modulated fiber (mode scrambler) such that theoptical intensity from a multimode beam is made uniform over the wholeillumination footprint. The shape of the fiber may be adjusted, forexample to be square or rectangular, such that the shape of theillumination can be matched to the shape of the data collection device(e.g., a CCD with square pixels). Also, in certain embodiments, a singlelaser excites two fluorophores, one with a narrow emission filter nearthe wavelength, and one with a wider band emission filter at longerwavelength. Such arrangement normalizes the relative intensities of thetwo dyes (with the same bandwidth filters, the dye further from thelaser wavelength would be much weaker). The embodiments herein also cancomprise a moving stage such that the chemistry (which requires heatingand cooling) can happen on the same instrument, but out of the opticaltrain. The systems herein also often contain an autofocus system toallow automated imaging of many tiles, and contain a fluidics system forperforming on-line fluidic changes. The individual components of thesystem/device (e.g., light source, camera, etc.) can optionally eachhave its own power source or supply or can optionally all be powered viaone source. As will be appreciated, while the components herein areoften described in isolation or in relation to only one or two othercomponents, that the various components in the embodiments are typicallyoperably and/or functionally connected and work together in thesystems/devices herein.

Flowcells

In various embodiments, the systems herein comprise one or moresubstrates upon which the nucleic acids to be sequenced are bound,attached or associated. See, e.g., WO 9844151 or WO0246456. In certainembodiments, the substrate is within a channel or other area as part ofa “flowcell.” The flowcells used in the various embodiments of theinvention can comprise millions of individual nucleic acid clusters,e.g., about 2-8 million clusters per channel. Each of such clusters cangive read lengths of at least 25 bases for DNA sequencing and 20 basesfor gene expression analysis. The systems herein can generate a gigabase(one billion bases) of sequence per run (e.g., 5 million nucleic acidclusters per channel, 8 channels per flowcell, 25 bases perpolynucleotide).

FIGS. 4A and 48 display one exemplary embodiment of a flowcell. As canbe seen, the particular flowcell embodiment, flowcell 400, comprisesbase layer 410 (e.g., of bomsilicate glass 1000 μm in depth), channellayer 420 (e.g., of etched silicon 100 μm in depth) overlaid upon thebase layer, and cover, or top, layer 430 (e.g., 300 μm in depth). Whenthe layers are assembled together, enclosed channels are formed havinginlet/outlets at either end through the cover. As will be apparent fromthe description of additional embodiments below, some flowcells cancomprise openings for the channels on the bottom of the flowcell.

The channeled layer can optionally be constructed using standardphotolithographic methods, with which those of skill in the art will befamiliar. One such method which can be used in the current invention,involves exposing a 100 μm layer of silicon and etching away the exposedchannel using Deep Reactive Ion Etching or wet etching.

It will be appreciated that while particular flowcell configurations arepresent herein, such configurations should not necessarily be taken aslimiting. Thus, for example, various flowcells herein can comprisedifferent numbers of channels (e.g., 1 channel, 2 or more channels, 4 ormore channels, or 6, 8, 10, 16 or more channels, etc. Additionally,various flowcells can comprise channels of different depths and/orwidths (different both between channels in different flowcells anddifferent between channels within the same flowcell). For example, whilethe channels formed in the cell in FIG. 4B are 100 μm deep, otherembodiments can optionally comprise channels of greater depth (e.g., 500μm) or lesser depth (e.g., 50 μm). Additional exemplary flowcell designsare shown in FIGS. 4C and 4D (e.g., a flowcell with “wide” channels,such as channels 440 in FIG. 4C, having two channels with 8 inlet andoutlet ports (ports 445—8 inlet and 8 outlet) to maintain flowuniformity and a center wall, such as wall 450, for added structuralsupport; or a flowcell with offset channels, such as the 16 offsetchannels (channels 480), etc.). The flowcells can be designed tomaximize the collection of fluorescence from the illuminated surface andobtain diffraction limited imaging. For example, in the design shown inFIG. 4C, in particular embodiments, the light comes into the channelthrough 1000 μm thick bottom layer 460, which can be made ofborosilicate glass, fused silica or other material as described herein,and the emitted light travels through 100 μm depth of aqueous solutionwithin the channel and 300 μm depth of “top” layer material 470.However, in some embodiments, the thickness of the “top” layer may beless than 300 μm to prevent spherical aberrations and to image adiffraction limited spot. For example the thickness of the top layer canbe around 170 μm for use with a standard diffraction limited opticalsystem. To use the thicker top layer without suffering from sphericalaberrations, the objective can optionally be custom designed, e.g., asdescribed herein.

In the various embodiments herein, the flowcells can be createdfrom/with a number of possible materials. For example, in someembodiments, the flowcells can comprise photosensitive glass(es) such asFoturan® (Mikroglas, Mainz, Germany) or Fotoform® (Hoya, Tokyo, Japan)that can be formed and manipulated as necessary. Other possiblematerials can include plastics such as cyclic olefin copolymers (e.g.,Topas® (Ticona, Florence, Ky.) or Zeonor® (Zeon Chemicals. Louisville,Ky.) which have excellent optical properties and can withstand elevatedtemperatures if need be (e.g., up to 100° C.). As will be apparent fromFIG. 4, the flowcells can comprise a number of different materialswithin the same cell. Thus, in some embodiments, the base layer, thewalls of the channels, and the top/cover layer can optionally be ofdifferent materials.

While the example in FIG. 4B shows a flowcell comprised of 3 layers,other embodiments can comprise 2 layers, e.g., a base layer havingchannels etched/ablated/formed within it and a top cover layer, etc.Additionally, other embodiments can comprise flowcells having only onelayer which comprises the flow channel etched/ablated/otherwise formedwithin it.

In some embodiments, the flowcells comprise Foturan®. Foturan is aphotosensitive glass which can be structured for a variety of purposes.It combines various desired glass properties (e.g., transparency,hardness, chemical and thermal resistance, etc.) and the ability toachieve very fine structures with tight tolerances and high aspectratios (hole depth/hole width). With Foturan® the smallest structurespossible are usually, e.g., 25 μm with a roughness of 1 μm.

FIG. 5A, gives a schematic diagram of one possible way of patterning aflowcell (e.g., one comprising Foturan®). First the desired pattern ismasked out with masks 500, onto the surface of substrate 510 which isthen exposed to UV light. In such exposure step, the glass is exposed toUV light at a wavelength between 290 and 330 nm. It can be possible toilluminate material thicknesses of up to 2 mm. An energy density ofapproximately 20 J/cm² is typically sufficient to structurize a 1 mmthick Foturan® plate. During the UV exposure step, silver or other dopedatoms are coalesced in the illuminated areas (areas 520). Next, during aheat treatment between 500° C. and 600° C., the glass crystallizesaround the silver atoms in area 520. Finally, the crystalline regions,when etched with a 10% hydrofluoric acid solution at room temperature(anisotropic etching), have an etching rate up to 20 times higher thanthat of the vitreous regions, thus resulting in channels 530. If wetchemical etching is supported by ultrasonic etching or by spray-etching,the resulting structures display a large aspect ratio. FIG. 5B shows atransmission spectra from a sample of Foturan glass (d=1 mm).

FIG. 6, panels A through E show an exemplary etching process toconstruct a sample flowcell as used herein. In FIG. 6A, channels 600(seen in an end view) and through-holes 605 (seen in an end view) areexposed/etched into layer 630. Layer 630 is the “top” layer of a twolayer flowcell as can be seen in FIG. 6E (mated with bottom layer 620).The through-holes (where reagents/fluids enter into the flowcellchannels) and channels can be etched into layer 630 through a 3-Dprocess such as those available from Invenios (Santa Barbara, Calif.).Top layer 630 can comprise Foturan which, as described, can be UVetched. Foturan, when exposed to UV, changes color and becomes opticallyopaque (or pseudo-opaque). Thus in FIG. 6B, layer 630 has been maskedand light exposed to produce darkened areas 610 within the layer(similar to the masking in FIG. 5A, but without the further etching).Such optically opaque areas can be helpful in blocking misdirectedlight, light scatter, or other nondesirable reflections that couldotherwise negatively affect the quality of sequence reading herein. Inother embodiments, a thin (e.g., 100-500 nm) layer of metal such aschrome or nickel is optionally deposited between the layers of theflowcell (e.g., between the top and bottom layers in FIG. 6E) to helpblock unwanted light scattering. FIGS. 6C and 6D display the mating ofbottom layer 620 with channel layer 630 and FIG. 6E shows a cut awayview of the same.

In various embodiments, the layers of the flowcells are attached to oneanother in any of a number of different ways. For example, the layerscan be attached via adhesives, bonding (e.g., heat, chemical, etc.),and/or mechanical methods. Those of skill in the art will be familiarwith numerous methods and techniques to attach variousglass/plastic/silicon layers to one another.

Again, while particular flowcell designs and constructions are describedherein, such descriptions should not necessarily be taken as limiting;other flowcells of the invention can comprise different materials anddesigns than those presented herein and/or can be created throughdifferent etching/ablation techniques or other creation methods thanthose disclosed herein. Thus, particular flowcell compositions orconstruction methods should not necessarily be taken as limiting on allembodiments.

Fluid Flow

In the various embodiments herein, the reagents, buffers, etc. used inthe sequencing of the nucleic acids are regulated and dispensed via afluid flow subsystem or aspect. FIGS. 7A-C present generalized diagramsof exemplary fluid flow arrangements of the invention, set up in one waypush, eight way pull, and one way pull configurations respectively. Ingeneral, the fluid flow subsystem transports the appropriate reagents(e.g., enzymes, buffers, dyes, nucleotides, etc.) at the appropriaterate and optionally at the appropriate temperature, from reagent storageareas (e.g., bottles, or other storage containers) through the flowcelland optionally to a waste receiving area.

The fluid flow aspect is optionally computer controlled and canoptionally control the temperature of the various reagent components.For example, certain components are optionally held at cooledtemperatures such as 4° C.+/−1° C. (e.g., for enzyme containingsolutions), while other reagents are optionally held at elevatedtemperatures (e.g., buffers to be flowed through the flowcell when aparticular enzymatic reaction is occurring at the elevated temperature).

In some embodiments, various solutions are optionally mixed prior toflow through the flowcell (e.g., a concentrated buffer mixed with adiluent, appropriate nucleotides, etc.). Such mixing and regulation isalso optionally controlled by the fluid flow aspect of the invention. Itis advantageous if the distance between the mixed fluids and theflowcell is minimized in many embodiments. Therefore the pump can beplaced after the flowcell and used to pull the reagents into theflowcell (FIGS. 7B and 7C) as opposed to having the pump push thereagents into the flowcell (as in FIG. 7A). Such pull configurationsmean that any materials trapped in dead volumes within the pump do notcontaminate the flowcell. The pump can be a syringe type pump, and canbe configured to have one syringe per flow channel to ensure even flowthrough each channel of the flowcell. The pump can be an 8 way pump, ifit is desired to use an 8 way flowcell, such as for example a Kloehn 8way syringe pump (Kloehn, Las Vegas, Nev.). A fluidics diagram of an 8way pull configuration is shown in FIG. 7B. In FIG. 7A, fluidic reagentsare stored in reagent containers 700 (e.g., buffers at room temperature,5× SSC buffer, enzymology buffer, water, cleavage buffer, etc.) and 710(e.g., cooled containers for enzymes, enzyme mixes, water, scanning mix,etc.). Pump 730 moves the fluids from the reagent containers throughreagent valve 740, priming/waste valve 770 and into/through flowcell760.

In FIG. 7B, fluidic reagents are stored in reagent containers 702 (e.g.,buffers at room temperature similar to those listed above) and 703(e.g., cooled containers for enzymes, etc. similar to those listedabove), linked through reagent valve 701. Those of skill in the art willbe familiar with multi-way valves (such as the reagent valves) used toallow controllable access of/to multiple lines/containers. The reagentvalve is linked into flowcell 705 via an optional priming valve (orwaste valve) 704, connected to optional priming pump 706. The primingpump can optionally draw reagents from the containers up through thetubing so that the reagents are “ready to go” into the flowcell. Thus,dead air, reagents at the wrong temperature (e.g., because of sitting intubing), etc. will be avoided. When the priming pump is drawing, theoutflow is shunted into the waste area. During non-priming use, thereagents can be pulled through the flowcell using 8 channel pump 707,which is connect to waste reservoir 708.

In either embodiment (push or pull), the fluidic configurations cancomprise “sipper” tubes or the like that extend into the various reagentcontainers in order to extract the reagents from the containers. FIG. 7Cshows a single channel pump rather than an 8 channel pump. Singlechannel pump 726 can also act as the optional priming pump, and thusoptional priming pump or waste valve 723 can be connected directly topump 726 through bypass 725. The arrangement of components is similar inthis embodiment as to that of FIG. 7B. Thus it comprises reagentcontainers 721 and 722, multi-way selector valve 720, flowcell 724, etc.

The fluid flow itself is optionally driven by any of a number of pumptypes, (e.g., positive/negative displacement, vacuum, peristaltic, etc.)such as an Encynova® 2-1 Pump (Encynova, Greeley, Colo.) or a Kloehn® V3Syringe Pump (Kloehn, Las Vegas, Nev.). Again, it will be appreciatedthat specific recitation of particular pumps, etc. herein should not betaken as necessarily limiting and that various embodiments can comprisedifferent pumps and/or pump types than those listed herein. In certainembodiments, the fluid delivery rate is from about 50 μL to about 500μL/min (e.g., controlled ±2 μL) for the 8 channels. In the 8 way pullconfiguration, the flow can be between 10-100 μl/min/channel, dependingon the process. In some embodiments, the maximum volume of nucleotidereagents required for sequencing a polynucleotide of 25 bases is about12 mL.

Which ever pump/pump type is used herein, the reagents are optionallytransported from their storage areas to the flowcell through tubing.Such tubing, such as PTFE, can be chosen in order to, e.g., minimizeinteraction with the reagents. The diameter of the tubing can varybetween embodiments (and/or optionally between different reagent storageareas), but can be chosen based on, e.g., the desire to decrease “deadvolume” or the amount of fluid left in the lines. Furthermore, the sizeof the tubing can optionally vary from one area of a flow path toanother. For example, the tube size from a reagent storage area can beof a different diameter than the size of the tube from the pump to theflowcell, etc.

The fluid flow subsystem of the invention also can control the flow rateof the reagents involved. The flow rate is optionally adjustable foreach flow path (e.g., some flow paths can proceed at higher flow ratesthan others; flow rates can optionally be reversed; different channelscan receive different reagent flows or different timings of reagentflows, etc.). The flow rate can be set in conjunction with the tubediameter for each flow path in order to have the proper volume ofreagent, etc in the flowcell at a given time. For example, in someembodiments, the tubing through which the reagents flow is 0.3 mm 1D,0.5 mm, or 1.0 mm while the flow rate is 480 μL/min or 120 μL/min. Insome embodiments, the speed of flow is optionally balanced to optimizethe reactions of interest. High flow can cause efficient clearing of thelines and minimize the time spent in changing the reagents in a givenflowcell volume, but can also cause a higher level of shear flow at thesubstrate surface and can cause a greater problem with leaks or bubbles.A typical flow rate for the introduction of reagents can be 15μl/min/channel in some embodiments.

The system can be further equipped with pressure sensors thatautomatically detect and report features of the fluidic performance ofthe system, such as leaks, blockages and flow volumes. Such pressure orflow sensors can be useful in instrument maintenance andtroubleshooting. The fluidic system can be controlled by the one or morecomputer component, e.g., as described below. It will be appreciatedthat the fluid flow configurations in the various embodiments of theinvention can vary, e.g., in terms of number of reagent containers,tubing length/diameter/composition, types of selector valves and pumps,etc.

Heating/Cooling

In some embodiments, the systems herein comprise a heating/coolingcontrol component having heating/cooling capabilities, e.g., throughPeltier devices, etc. Optionally, the various components herein (e.g.,the flowcell and its contents) can be heated by a resistive heatingelement and cooled through convection to create reaction conditionsabove ambient temperature. Such heating/cooling component(s) can controlthe temperature of the flowcells (and the fluids within them) during thevarious reactions required in sequencing-by-synthesis. An exemplaryflowcell temperature control system is shown in FIG. 8 (in isolationfrom the other components of the system). In FIG. 8, Peltier fan 800 isshown in relationship to heat sink 810 and Peltier heater 820. Theflowcell heating/cooling component is optionally positionable and/ormovable in relation to the other components of the system (e.g., theflowcell and flowcell holder, etc.). Thus, the heating/cooling componentcan be moved into place when needed (e.g., to raise the temperature ofthe reagents in the flowcell to allow for enzyme activity, etc.) andmoved away when not needed. Additionally and/or alternatively, theflowcell and flowcell holder can optionally be moved in relation to theheating/cooling component. See FIG. 10A and 10B below. In variousembodiments, the temperature control elements control the flowcelltemperature, e.g., from about 20° C. to about 60° C. or any othertemperature/temperature ranges as required by the reactions to be donewithin the systems/devices. The temperature of the heating element canbe adjusted to control the temperature of the flowcell and the reagentstherein. As the flowcell is exposed to a flow of cooled reagents, thetemperature of the heating element may be higher than the temperaturedesired at the surface of the flowcell. For example the heating elementmay be set to 55° C. to obtain a flowcell temperature of 45° C.

Those of skill in the art will be familiar with Peltier devices used fortemperature control (which can optionally be used in the systemsherein). Again, it will be appreciated that while certainheating/cooling devices are recited herein, such should not be construedas necessarily limiting. Thus, in certain embodiments heating/coolingdevices other than Peltier devices are optionally comprised within thepresent invention. In typical embodiments, notwithstanding the type ofdevice, the heating/cooling component is optionally controlled (e.g., interms of temperature, time at particular temperatures, movement of thecomponent, and/or movement of other devices such as the flowcell holderto the heating/cooling component) by the computer component (see below).

In some embodiments, additional heating/cooling elements can optionallyregulate the temperature of other components in addition to or alternateto the flowed. For example, heating/cooling components can optionallyregulate the temperature of the camera, the reagent reservoirs, whichcan be cooled, for example to 4° C. to prolong the storage life of thereagents during long sequencing runs, the temperature of the atmosphereinside the instrument etc.

Multiple Flowcells and Alternative TIRF and Heating/Cooling Approaches

In certain embodiments herein, the systems/devices can compriseadditional approaches to flowcell configuration, TIR illumination,heating/cooling configurations of the flowcell(s), and in how theflowcells are held/stabilized within the device. While such approachescan optionally be utilized together in certain embodiments, it will beappreciated that they each can be used in any combination, e.g., witheach other, with any of the other approaches described herein, etc.

In some embodiments, the flowcells herein can be “bottom flow”flowcells. Thus, as opposed to the flowcells, e.g., as shown in FIGS. 4,6, and 9 where the flowcells are clamped down and fluid flow enters fromthe top side of the flowcell, some flowcells can comprise configurationsthat allow fluid flow that enters from the bottom of the flowcell. Suchbottom flowcells can be similar in construction and composition as “topflow” flowcells. In some embodiments bottom flow flowcells can compriseless fluidic dead volume (and use more of the whole channel length thantop flow flowcells, e.g., since the ends of the flowcells are notcovered by clamps/manifolds, etc.). See, e.g., FIG. 44-49.

Bottom flow flowcells can optionally be held to the flowcell holderthrough vacuum chucking rather than clamps. Thus, a vacuum can hold theflowcell into the correct position within the device so that properillumination and imaging can take place. Cf., FIGS. 44-49 with FIG. 9.Thus, some embodiments herein also comprise one or more vacuum creationdevice to create a vacuum (or partial vacuum, etc.) to hold the flowcelland/or prism to the flowcell holder, XY stage, etc.

Various examples of flowcell holder manifolds are shown in FIG. 46 thatcan be used with bottom flow flowcells. As can be seen, the fluidsflowed into/through/out of the flowcell are directed through variousbranching tubes within the manifolds to/from specific channels withinthe flowcell. Again, such embodiments can optionally not obstruct any(or not substantially any) of the top surface of the flowcell whichmight interfere with illumination/imaging of the MI length of thechannels. FIG. 47 displays an exemplary fluidic valve. Such valve has nomoving parts or vibrations and a low dead volume. In such arrangements,each reagent bottle/container can have an open/close valve. Afterdrawing a fluid, air can be injected before closing the reservoir valvethereby forcing an air gap valve between reagents. Cooled reagents canbe returned to their reservoirs and all reagents in case of a systemshut down. Also, an air injection pump can be added to the push/pullpump (e.g., a kloehn pump).

Another approach to illumination can comprise “top down” illumination.Such top down approach can be useful when used in conjunction withvacuum chucking (and bottom temperature control below). It canoptionally be problematic to illuminate from the bottom (e.g., as inFIG. 1, etc.) in configurations with vacuum chucking and bottomtemperature control since such embodiments often utilize the space belowthe flowcell. As can be seen in FIG. 44, top down or side illuminationcomes from above into prism 4401 upon which flowcell 4402 rests (and isoptionally held down by vacuum). Such arrangement can also help preventbowing of the flowcell which presentation can aid in auto focusing andflat field imaging and can aid in configuration with multiple flowcellshaving simultaneous reading, etc. Laser illumination 4400 is also shownentering into the prism in FIG. 44 as is mirror 4405 andmanifold/fluidic connector 4404.

FIG. 45 shows another approach to thermodynamic control of a flowcell(and the reagents and reactions within it). FIG. 45 shows an exemplaryembodiment of a bottom temperature controlled device. In some suchembodiments, the aspect can comprise a water cooled bench that can helpassure dimensional stability during read cycles and controlled scanbuffer temperature. A thermal plate can extend past the prism andflowcell and under the manifolds to optionally help in uniformtemperature control. Fluids can optionally be preheated when passingthrough the inlet manifold. Also, RTD temperature feedback can beimbedded in top of the prism to assure that the flowcell is at thedesired set temperature and that thermal resistant effects of the prismare minimized.

Configurations having multiple flowcells within a flowcell holder areshown in FIG. 48. As can be seen, up to four flowcells can be loadedinto the holder in FIG. 48A (or two double wide flowcells, e.g., having18-20 channels each). Peltiers or other similar devices can be beneaththe flowcells and can optionally be water cooled through the holderbench aspect (which can be kept at room temperature optionally).

Stage and Flowcell Holder

Placement and movement of the flowcell (and thus the nucleic acids to besequenced) is controlled and secured by, e.g., a movable stage uponwhich the flowcell and flowcell holder (or other substrate) are located.Such movable stage can optionally allow movement of the flowcell inrelation to the laser illumination and lens objective to read thesequencing reactions within the channels. If desired, the scanning stageor other components can be actively cooled during the scanning cycle tocontrol the temperature of the substrate during the imaging cycles.

FIG. 9, panels A through D, displays schematic diagrams of an exemplaryflowcell holder of the current system. FIG. 9A shows flowcell holder 900before a flowcell is placed upon it. As can be seen, the holdercomprises adjustable clamps 910 (optionally spring loaded) to securelyfasten the flowcell to the holder and optionally one or more manifolds(e.g., optionally comprised within the clamps) to fluidically connectthe flowcell channels to the rest of the fluidic system. A manifold canindividually connect each of the channels in parallel. Alternatively, amanifold can connect the channels such that they are connected via asingle inlet line that is split to flow in parallel to each channel, orcan be configured as a “serpentine” configuration to make a single fluidflow. Such a manifold can be configured to contain a single 1-8 split,or can comprise a binary splitter wherein each fluid channel is onlysplit into 2, to obtain a split from 1-2-4-8, in order to give a moreuniform flow along each of the 8 channels. In the 8 way pullconfiguration, the “exit” manifold from the flowcell can comprise 8individual ports, each connected to a barrel of an 8 way syringe pump,whilst the “inlet” manifold can contain a single entry tube to reducethe length of tubing needed to till the flowcell. The inlet manifold cancontain a 1-8 splitter or a binary 1-2-4-8 splitter for partitioning theflow evenly down each of the 8 channels. FIG. 9B also shows the presenceof adjustable prism 920 that optionally can be raised/lowered to comeinto contact with the underside of the flowcell. The prism is used inconjunction with the lasers in the TIRF activity. In particularembodiments, oil (e.g., immersion oil such as that available fromCargille; catalog #19570 or the like) is placed between the prism andthe flowcell in a uniform and continuous layer to create total internalreflection through the layer of air between the prism and the flowcellglass. FIG. 9C shows placement of flowcell 930 upon the holder and prismand FIG. 9D shows the flowcell clamped to the flowcell holder withhandle/clamp 940 being lowered to help secure the clamps and flowcell.

The flowcell and flowcell holder can be situated upon a movable stage orplatform. Such stage optionally is adjustable along, X, Y, and Z axes.This allows fine scale height and placement adjustment of the flowcellin relation to the lasers, camera, lens optics, etc, and allows thesurface of the flowcell to be kept in focus relative to the imagingdevice. Furthermore, the movable stage can optionally allow the flowcellto be moved back and forth between the heating/cooling component and theoptic/laser components (i.e., to allow enzymatic reactions when heatedand to quantify the outcome of such reactions with the camera/lasercomponents). FIG. 10 shows photographs depicting movement of flowcell1020 and flowcell holder 1010 between the heating/cooling element (leftpicture) and the camera/laser elements (right picture). Thus the x and ycomponents can allow the flowcell to be moved laterally (e.g., by 10s ofcentimeters), whilst the height can be adjusted (e.g., by 10s ofnanometers) vertically to allow focusing of the images. Alternatively,the stage also can be simply an XY stage with no vertical setting, andthe lens objective can be adjustable in the Z plane to ensure focus ismaintained. It will be appreciated that the heating/cooling elements areoptionally movable as well, e.g., in order to come into closer proximitywith the flowcell, etc. Cf., FIG. 10 left picture (heating/coolingdevice raised) and FIG. 10 right picture (heating/cooling device loweredonto flowcell).

FIG. 10A shows a photograph of the instrument before and during theheating step. Peltier device 1000 (comprised of fan 1001, heat sink 1002and heater unit 1003) moves in the vertical direction to come intocontact with the flowcell 1020 and flowcell holder 1010 mounted on XYstage 1050. Reagents are introduced into the flowcell via tube 1040. Theflowcell can move to a position located under camera 1030 for imaging. Aschematic representation of the device in the imaging location is shownin FIG. 10B, where the Peltier device 1070 is in the raised position(with fan 1071, heatsink 1072, and heater 1073), flowcell 1085 and stage1086 are sited next to the fiber optic mount 1090 and below lensobjective 1080. The fiber optic mount is connected to the Z stage 1075,which also controls the height of lens objective 1080. The flowcell isclamped in place onto the flowcell holder by the manifold lever/handle1095.

Additionally, it will be appreciated that the various components herein,e.g., the laser components, heating/cooling components, etc., aretypically arranged on a scaffolding, chassis, or framework andoptionally enclosed within a housing to fully or partially enclose theinstrument. The particular configuration of such framework and/orhousing can optionally vary in different embodiments based upon, e.g.,the particular components, their size, etc. In typical embodimentshowever, the framework keeps the various components secure and in theproper location and orientation while also optionally aiding in themovement of the components when necessary. The framework should be rigidenough to prevent vibrations within the instrument and the variouscomponents. For example the mode scrambler can be motion damped andvibrationally isolated from the stage to prevent shaking of the flowcellduring imaging. FIG. 11A shows a schematic displaying an exemplaryframework holding the camera (1100), heating/cooling components 1110,(cf., FIG. 8) flowcell and flowcell holder, and movable stage 1120.Additional aspects of framework and mounting that aid in tying togetherthe various components and aspects of the device/system include variousalignment and mounting pins/locations can be seen in FIG. 11B whichshows the bearing slide for laser piece vertical adjustment 1165 andflowcell leveling adjustment component 1175. Other frameworks andhousing, including external covers (skins) for the housing can be seenin FIG. 12 along with computer monitor 1201.

Excitation and Observation

In certain embodiments herein, the incorporation of specific nucleicacid bases with their accompanying specific fluorescences is tracked vialaser excitation and camera observation. In various embodiments, theillumination is performed using Total Internal Reflection (TIR)comprising a laser component. It will be appreciated that a “TIRFlaser,” “TIRF laser system,” “TIR laser.” and other similar terminologyherein refers to a TIRF (Total Internal Reflection Fluorescence) baseddetection instrument/system using excitation, e.g., lasers or othertypes of non-laser excitation from such light sources as LED, halogen,and xenon arc lamps (all of which are also included in the currentdescription of TIRF, TIRF laser, TIRF laser system, etc. herein). Thus,a “TIRF laser” is a laser used with a TIRF system, while a “TIRF lasersystem” is a TIRF system using a laser, etc. Again, however, the TIRFsystems herein (even when described in terms of having laser usage,etc.) should also be understood to include those TIRFsystems/instruments comprising non-laser based excitation sources. Thoseof skill in the art will be well aware of different aspects of TIRFsystems and their general use. In various embodiments, the cameracomponent comprises a CCD camera. In some embodiments, the lasercomprises dual individually modulated 50 mW to 500 mW solid state and/orsemiconductor lasers coupled to a TIRF prism, optionally with excitationwavelengths of 532 nm and 660 nm. The coupling of the laser into theinstrument can be via an optical fiber to help ensure that thefootprints of the two lasers are focused on the same area of thesubstrate (i.e., overlap).

Mode Scrambling

In the various embodiments herein, the area wherein the laser(s) orother excitation source(s) illuminate the sample (the area of whichillumination is referred to as the “footprint”) is typically desired tobe spatially flat and uniform. In many embodiments the devices/systemsherein take advantage of properties of multimode fibers that allowpropagation of all optical modes through their cores with near equalamplitude to produce a flat or top-hat profile illumination footprintfrom the laser on the illuminated substrate surface (e.g., the surfaceof a flowcell), etc. However, the finite number of modes present in suchfibers can constructively and destructively interfere with each otherand produce local minima and maxima in the intensity profile of thelaser (or other light). See, e.g., FIGS. 33A and 34A which showminima/maxima resulting from uncorrected output from multimode fibers.To ameliorate this problem, some embodiments herein produce asubstantially uniform footprint by use of dynamic mode scrambling byconstantly changing the index of refraction within the illuminationbeam, e.g., by modulating the beam with a waveplate, or by shaking,squeezing or compressing one or more areas of a fiber carrying theillumination beam. Thus, some embodiments of the current inventionproduce a substantially uniform flat-top output (i.e., a substantiallyuniform illumination/excitation footprint from a laser or light source)by dynamically scrambling the modes in an illuminating beam, e.g., bysqueezing/compressing a fiber carrying the beam in one or more area overits length. FIGS. 33 and 34 summarize various embodiments of modescrambling as described herein. See below.

Dynamic Mode Scrambling and Low Loss Beam Shaping

In certain embodiments, the devices herein comprise component(s) toproduce a “top-hat” illumination, e.g., a uniform or substantiallyuniform illumination over a particular illumination footprint, as seenin FIG. 35. Such embodiments comprise one or more aspects thatdynamically change the index of refraction within the mediumtransmitting the illumination (e.g., a fiber) at one or more nodes. Forexample, a fiber can be squeezed at various locations along its lengthto induce a continuously changing index of refraction. Such squeezing ofthe fiber, e.g., a Step Index Fiber, can be used to spatially/temporallyscramble the modes in the fiber to cause sufficient overlap over adesired integration time of the output illumination. As explained alsoherein (see below) the fiber can also be shaken, rotated, vibrated orphysically deformed in other ways to change the optical path through thefiber.

In general, the dynamic scrambling of the modes in the fibers allowsachievement of spatially uniform illumination over a minimum userdefined integration time. This thus prevents interference of propagatingmodes of monochromatic light in multimode fibers which would producelight and dark patterns in the resulting beam. It is optionallysufficient that these modes disappear over the minimum integration time.Thus, in some embodiments, the relative path lengths of these modeswithin the illumination beam are rapidly varied by introducing timevariable curvature and index variations into the fiber, e.g., bymechanical means.

It will be appreciated that several parameters of the dynamic modescrambling can optionally be varied or can comprise a range of differentconfigurations. However, in general, dynamic mode scrambling comprisesone or more aspects/components used to dynamically change the index ofrefraction of an illumination beam in order to average out an endillumination footprint. While many existing refractive optical conceptsrequire an input Gaussian beam and existing diffractive optical conceptsare often wavelength dependent, the present embodiment does not requirea Gaussian beam input and is wavelength independent.

In their various embodiments, the devices/systems herein desire auniformly illuminated field for excitation/measurement of the sequencingreactions, etc. Thus, the uneven light/dark patterns that result frominterference of propagating modes of monochromatic light in a multimodefiber is typically undesirable. Averaging of the light output over anillumination footprint (over a period of observation time such as thetime captured by a camera during an imaging) to allow integration of thelight means that the light/dark patterns “disappear” or are averagedout, and thus the excitation intensity seen by each fluorophore on thesurface should be uniform.

Underlying dynamic mode scrambling, is the constant varying of the indexof refraction at a point or node of the light beam over time (e.g., byphysically squeezing a fiber over time) which causes the light to bescrambled and take different paths and thus averages out the lightoutput in the illumination footprint. Thus, the position of interferenceminima and maxima changes as the index of refraction of the input beamis changed. If the index of refraction is changed at a frequency that isfaster than the image acquisition time, then a spatially uniform imagecan be produced in the timescale of the observation.

It will be appreciated that the current embodiment should not beconfused with the common usage of “mode scramble” which most oftenrefers to randomization of an input mode or modes relative to theoutput. The desired function of the current embodiment is to temporallyas well as spatially randomize modes, i.e., producing dynamicscrambling.

The dynamic mode scrambling of the current embodiment can also be usedin conjunction with fibers comprising cores of particular shapes toachieve a beam shape with uniform illumination. For example, squeezing afiber with a square core will result in a uniformly illuminated squarebeam. The beam can be shaped along a particular axis to make arectangle, or oval shape, which beam is imaged as square or circularwhen it hits upon the imaging surface. See FIGS. 17-18. For example,rectangular beams can be generated from optical fibers, as shown in FIG.34.

FIG. 35 shows the optical output from a variety of different lasers,fibers, and mode scrambling aspects, etc. During device operation, theends of the fibers were re-imaged onto a beam profiler. FIG. 35 showsthe effect of dynamic modescrambling (i.e., by manipulation of thefibers at one or more nodes with, e.g., piezo-electric actuators) bycomparing the images from different wavelength lasers (e.g., 532 nm and550 nm) and laser times (solid state and diode) in conjunction withdifferent beam shapers (two versions of rectangles and a circle) byshowing the output when the dynamic modescrambling is “on” versus thelight output when the modescrambling is “off” for each laser type, etc.

It will be appreciated that one embodiment of the device can thereforecomprise a dynamic mode scrambler as opposed to static mode scrambler.It is the dynamic variation of index of refraction that causes the modesto overlap over the desired integration time. The index of refraction isconstantly changed at one or more location (node). For example, a fibertransmitting the illumination is constantly squeezed at a point with achanging degree of intensity (e.g., from no squeezing to maximumsqueezing and back again). The fiber can be temporarily deformed by suchsqueezing so that its shape changes from a circle to an ellipse to acircle, etc. which, in turn, keeps changing the index of refraction. Assoon as the squeezing stops, the mode scrambling stops.

Efficiency of averaging of the illumination output in a footprintdepends on length of image capture, the degree of change in index ofrefraction, the type/strength of the light source, etc. Thus, it is auser controllable variable and should not necessarily be taken aslimiting. The user can optionally control the degree of scrambling tofine tune the averaging of light output in a footprint.

Thus, the time period over which light output averaging is measured isvariable, e.g., it can be the period during which an image is capturedof the area illuminated by the light output (e.g., tiles (specific imagecapture areas) upon the flowcells in certain sequencing embodimentsherein). In certain embodiments, the time period of scramblingefficiency is equivalent to or substantially equivalent to the exposeperiod for each image captured by a camera (e.g., the CCD camera inparticular sequencing embodiments herein). It will be appreciated thatsuch exposure times can vary from embodiment to embodiment, e.g., fromless than 1 millisecond to over 1 hour or more depending upon theparticular requirements of the embodiment (e.g., at least 1, 5, 10, 25,50, 100, 250, 500 or more microseconds; at least 1, 5, 10, 25, 50, 100,250, 500 or more milliseconds; at least 1, 5, 10, 25, 50, 100, 250, 500or more seconds, etc.). For the sequencing reactions described herein,the imaging time may be of the order of 50-500 milliseconds perexposure.

In various embodiments, the current dynamic mode scrambler can, nomatter the overall system with which it is used, be used with differentlight sources/types, different beam media, different ways of changingthe index of refraction, different numbers of nodes where the index ofrefraction is changed, etc.

Dynamic mode scrambling is not limited by the particularlight/illumination used. Thus, for example, while many embodimentsherein optionally use lasers of particular wavelength (e.g., 532 and/or660 nm), other embodiments can use illumination of entirely differentwavelength. The lasers used with dynamic mode scrambling can be, e.g.,visible light lasers, IR lasers, narrow alignment lasers, broadlinewidth lasers, etc. Again, while particular laser wavelengths arementioned herein, such recitation should not necessarily be taken aslimiting. Of course, it will be appreciated with each different lasertype/strength used, that correspondingly, other parameters areoptionally adjusted to achieve substantially uniform illumination. Forexample, the number of nodes where the index of refraction is changedand/or the rate of change of the index at such nodes is optionallydifferent for different light sources to achieve the same degree ofuniformity of the footprint.

Also, while the examples herein are generally addressed in terms of modescrambling in fiber optic lines, dynamic mode scrambling is alsooptionally used with light transmitted through glass, plastic, non-fiberoptic lines, air, vacuum, etc. Thus, dynamic mode scrambling is notlimited by the medium in which the light is transmitted. Here too,differences in the transmission medium can optionally also match with adifference in other aspects of the mode scrambler needed to achievesubstantially uniform output. For example, for light transmitted throughair/vacuum (i.e., not contained within a fiber, etc.), the index ofrefraction is optionally changed/varied by changes in temperature ratherthan any mechanical change in the transport medium.

The index of refraction can optionally be varied through a number ofways. For example, as mentioned above, when the light is not transmittedthrough a cable/fiber, but rather traverses air/vacuum, the index ofrefraction of the light beam can be varied by changes in temperature.Thus, one or more heaters/coolers can he used to vary the temperature ofone or more node of the light beam to change the index of refraction.For beams that travel through a fiber/cable, the physical properties ofthe fiber can be changed in order to vary the index of refraction. Forexample, the fiber can be physically bent, shaken, twisted, squeezed,compressed, pulled, or heated/cooled at one or more nodes to change theindex of refraction at those points. The physical interaction with thefiber can be through actual mechanical manipulation (e.g., throughrollers, pinchers, etc. and/or through piezo-electric actuators thatsqueeze the fiber (e.g., similar to those available from GeneralPhotonics (Chino, Calif.)), etc.). Generally, any way of varying theindex of refraction can be used.

In addition to different ways of changing the index of refraction, therate of change of the index, the number of nodes, etc. are alsooptionally variable. Thus, in different embodiments, dynamic modescrambling can comprise one or more node (i.e., area where the index isvaried) on an illumination beam, which node can be fixed/static ormovable along the light beam. In a general, but not limiting sense, thegreater the number of nodes, the more scrambling occurs. Similarly, formultiple nodes it is typically preferred that the changes in refractionnot be synchronized with one another (i.e., it is preferred that thevariation in index of refraction be random).

FIGS. 33-35 show examples of mode scrambling with various fiber shapesand various light sources. As can be seen from the images, substantiallyuniform “top hat” illumination is achieved when the dynamic modescramble is performed using a vibrating or squeezed fiber. The figuresalso illustrate that images can be shaped through use of shaped-corefibers. FIG. 33 shows a nonscrambled beam output (A) compared with beamoutputs wherein the fiber was shaken, e.g., through use of a MKIII MSfrom Point Source (Hamble, UK) (B), vibrated, e.g., with an MKIV MS fromPoint Source (C), or squeezed, e.g., through use of one or morepiezo-electric squeezer/compressors (e.g., squeezed over 6 nodes atabout 500-600 Htz. per node) (D). The results shown in FIG. 33 were allperformed with the same fiber and laser types (e.g., 15 micron stepindex fiber and a 532 nm solid state laser). Similar results are shownin FIG. 34A-D for a rectangular core fiber: nonscrambled (A), shaken(B), vibrated (C), or squeezed (D). In FIG. 34 the examples were alldone with the same fiber/laser types. FIG. 35 shows similar results on anumber of different laser sources and scrambling procedures. Thus inFIG. 35 the panels correspond to: 660 um wavelength diode laser in arectangular core fiber with no mode scrambling (A) and the same fiberwith dynamic mode scrambling (B); a 532 um wavelength solid state laserwith no mode scrambling (C) and the same fiber with dynamic modescrambling (D); a 660 um wavelength diode laser (a second rectangular)with no mode scrambling (E) and the same fiber with dynamic modescrambling (F); a 532 solid state second rectangular fiber with noscrambling (G) and with dynamic mode scrambling (H); a 660 um diodelaser (round) with no mode scrambling (I) and with dynamic modescrambling (J); a 532 um solid state laser with no mode scrambling (K)and with dynamic mode scrambling (L).

Low Loss Beam Shapers

In some embodiments herein, specific beam shapes such as a square orrectangular laser beams are optionally used. Such shaped illuminationallows for efficient exposure and tiling over a surface, e.g.,comprising a nucleic acid sample, which can result in higher throughputin various devices herein. This can be advantageous in cases where theimaging is performed using a CCD device with square pixels, as theillumination footprint and imaging area can be tiled to preventillumination, and photobleaching of areas outside the image capturearea.

In some embodiments herein, instead of using a mask to shape the beamand re-image the mask onto the sample surface (which can optionallywaste energy outside of the mask), the laser is coupled into a square orrectangular (or other shaped) core fiber. Thus, all the available laserpower is efficiently used for illumination. Propagation down asufficient length of such shaped fiber fills the core efficiently toproduce the desired illumination shape. The end of this fiber can thenbe re-imaged onto a sample, e.g., a flowcell substrate. In particularembodiments, such re-imaging of the illumination from the fiber istypically desired to not substantially disturb the top-hat profileand/or beam shape achieved from scrambling and/or beam shaping (or evento distort the beam when it has not been beam shaped or scrambled).Thus, re-imaging aspects (e.g., lens(es), etc.) are appropriately chosento not distort the achieved profile and optionally to correctly magnifythe light output onto the flowcell, etc. Re-imaging, in particularembodiments, can also be chosen to be achromatic (i.e., to be able tofunction with any wavelength light). In some embodiments, re-imagingcomponents can also be “pistoned” by slightly moving the re-imagingcomponents to have the illumination land properly on particular areas ofthe flowcell.

Illumination uniformity in such embodiments can optionally be controlledby the condition of the beam launched into the shaped fiber coupled withthe length of the fiber. Illumination uniformity optionally can beenhanced by dynamically scrambling the modes within the shaped fiber.For example utilizing a device that continuously squeezes the shapedcore fiber at various locations. See above. The delivered beamdimensions at the sample surface optionally can be manipulated byimaging lenses.

FIGS. 34 and 35 show the results of use of a rectangular core opticalfiber. The end of the fiber was re-imaged onto a beam profiler. Theimage from the beam profile illustrates the desired rectangular beamwith uniform illumination in the vertical and horizontal dimensions.

The dynamic mode scrambling and/or beam shaping systems comprisecomponents to generate and deliver a substantially uniform andwavelength-switchable evanescent beam to the lower surface of a flowcellchannel (or other substrate) in an SBS reader instrument. As isapparent, these components interface with several othermodules/components in the overall SBS system (e.g., the various opticscomponents described above, etc.), and can be controlled/directedthrough one or more computer component.

Even though the current dynamic mode scrambling and beam shapingembodiments include, and are described throughout in terms of theirinteraction with, nucleic acid sequencing systems (e.g., varioussequencing by synthesis configurations as described herein), it will beappreciated by those of skill in the art that such embodiments are alsoapplicable to a wide range of other uses/systems. Thus, dynamic modescrambling can be included in myriad systems comprising one or moreaspects to dynamically vary the index of refraction of an illuminationbeam to mix the optic modes of a multimode optical fiber in order toproduce a substantially uniform image or output in a desired timeframe(e.g., such as during the image capture time for a camera or the like).Dynamic mode scrambling can optionally be utilized with systems such asthose tracking fluorescence on a plate or microarray or the like, i.e.,uses that do not comprise tracking of sequencing reactions.

Mode Scrambling Using Waveplates

In various aspects herein, the invention comprises a system for mixingoptic modes in a multimode optic fiber through use of waveplates. Suchsystems comprise a light source (e.g., a laser) which sends lightthrough a multimode optic fiber and also optionally through at least onewaveplate and then optionally through a re-imaging lens(es), prism, andonto a substrate (flowcell). The waveplates in such systems can comprise“rotating” waveplates. In some embodiments the waveplates actuallyphysically rotate at various rpms, while in other embodiments, such aswith liquid crystal waveplates, the plate “rotates” and alters thepolarization of the light passing through it by varying voltage acrossthe liquid crystal. In certain embodiments, the waveplate comprises twoor more sections of oriented retarders each of which rotatespolarization in different directions. In typical embodiments, the lightoutput from the fiber comprises a substantially uniform light output ona surface over a defined time period. The light output on the surfacesin various embodiments herein comprises reduced intensity minima andreduced intensity maxima in comparison to the output from a multimodeoptic fiber that does not comprise one or more rotating waveplates.

In other aspects, the invention comprises methods for equalizing lightoutput from a multimode optic fiber over a surface in a defined timeperiod by sending light from a light source (e.g., a laser) through amultimode optic fiber and through one or more rotating waveplates. Insome embodiments, the output on the surface comprises reduced intensityminima and reduced intensity maxima as compared to the output from amultimode optic fiber that does not comprise one or more rotatingwaveplate. In some embodiments the waveplates actually physically rotateat various rpms, while in other embodiments, such as with liquid crystalwaveplates, the plate “rotates” and alters the polarization of the lightpassing through it by varying the voltage across the liquid crystal. Incertain embodiments, the waveplate comprises two or more sections oforiented retarders each of which rotates polarization in differentdirections.

As used herein in some embodiments, a “waveplate” (or retardation plateor phase shifter or the like) refers to an optical device that altersvelocity of light rays as they pass through it, thus, creating a phasedifference. Waveplates are typically comprised of a birefringentcrystal. Some embodiments can comprise a liquid crystal waveplate.

As described above, in particular embodiments comprising laser or othersource excitation, the illumination of the sample (the area of whichillumination is referred to as the “footprint”) is spatially flat anduniform. The optic instruments herein exploit the properties ofmultimode fibers that allow propagation of all optical modes throughtheir core with near equal amplitude which produces a flat or top-hatprofile of the footprint. However the finite number of modes present insuch fibers can constructively and destructively interfere with eachother, thus producing local minima and maxima in the intensity profileof the laser (or other light). Some embodiments produce a uniformfootprint by physically shaking the fiber at a timescale shorter thanthe exposure time of the camera capturing the images, which averages theintensity minima and maxima and produces a uniform flat top footprint.This shaking can require an off balance DC motor that rotates and shakesthe fiber, which in some instances can cause undesired noise andvibrations that need to be damped to avoid causing imaging problems. Theshaking can also adversely affect reliability since off balance DCmotors have a shorter mean time between failure than balanced motors,and may increase physical wear on the fiber. Because of these factors,mode mixing in a multimode optical fiber without mechanical vibrationsand, in some instances without moving parts, by using waveplates can beadvantageous in some instances.

One embodiment of the current invention produces a substantially uniformflat-top beam (i.e., illumination/excitation area or footprint) bymixing the modes of the multimode optical fiber using a rotating λ/2waveplate (retarding plate). The spatial content of the modes depends onthe state of polarization of the input light. As polarization ischanged, the spatial content is changed. Thus, the position ofinterference minima and maxima changes as the polarization of the inputbeam is changed. If the waveplate is rotated at an angular frequencythat is faster than image acquisition time, then a spatially uniformimage can be produced in the timescale of the observation. Thus, inparticular embodiments, the waveplate completes one or more rotationduring a certain time period. The time period is, e.g., one during whichan image is captured of the area illuminated by the light output (e.g.,substrate areas of the flowcells in certain sequencing embodimentsherein). Thus, in certain embodiments, the time period is equivalent toor substantially equivalent to the expose period for each image capturedby a camera (e.g., a CCD camera in particular sequencing embodimentsherein). It will be appreciated that such exposure times can vary fromembodiment to embodiment, e.g., from less than 1 msec to over 1 hour ormore depending upon the particular requirements of the embodiment (e.g.,at least 1, 5, 10, 25, 50, 100, 250, 500 or more μsec; at least 1, 5,10, 25, 50, 100, 250, 500 or more msec; at least 1, 5, 10, 25, 50, 100,250, 500 or more seconds, etc.). For the cameras used herein, theexposure time may be 50-500 milliseconds. In certain embodiments thewaveplates can rotate less than or more than a full rotation during thetime period, thus, in some embodiments, aliasing can also be included.

While the rotation of the polarization can be accomplished by a numberof ways, typical embodiments rotate the waveplate. In particularembodiments herein, a λ/2 waveplate (see waveplate 3800 in FIG. 38) in asuitable housing is rotated by a suitable DC motor or the like,operating at a speed fast enough so that a spatially substantiallyuniform image is produced during the appropriate image capture time.Other embodiments comprise modified waveplate(s) which consist ofseveral sections of oriented waveplates or smaller pieces, with the fastaxis oriented in different directions (see waveplate 3900 in FIG. 39).Since sections rotate the polarization in different ways/amounts, a muchfaster mixing of the modes can occur and the DC motor optionally doesnot rotate as fast as in the embodiments with several sections. In yetother embodiments, other devices, such as liquid crystals (such as, butnot limited to, those manufactured by Meadowlark Optics (Frederick,Colo.)) can be used to rotate the polarization of the laser. With suchliquid crystals, the polarization can be rotated by varying the voltageacross the device.

FIG. 40 shows a schematic diagram representing an exemplary arrangementof an embodiment of the invention. In FIG. 40 linearly polarized light4100 (200 mW, 532 nm) from diode pumped solid state laser (4200) isattenuated by use of several OD filters. The intensity of the beam isfurther controlled by λ/2 waveplate 4900 (e.g., Casix, Fuzhou, Fujian,China) and polarizing beamsplitter cube 4300 (e.g., Thorlabs, Newton.N.J.). In various embodiments, the entire laser intensity is not needed,thus, in the example described only about 0.1 μW is used. Rotation ofthe waveplate 4900 allows a precise control of the input laser powerwhile keeping the polarization fixed. The beam is then passed throughsecond λ/2 retarding waveplate 4400 and steered by two mirrors 4500 and4600 into microscope objective 4700 (e.g., Nikon, 20×NA 0.3). Themicroscope objective reduces the beam to the required size to beaccepted by multimode optical fiber, 4800 200 μm core, 0.22NA (e.g., OZoptics, Ottawa, Canada). The output end of the fiber in the embodimentshown is placed directly on the chip of CCD camera 4905 (e.g., Cascade512, Photomctrics, Tucson, Ariz.). In the exemplary embodiment shown,the camera was operated in frame transfer mode and exposures of 100 mswere adequate to capture the beam profile as explained herein.

In various embodiments, the current invention, no matter the overallsystem with which it is used, can comprise different waveplates (e.g.,different in terms of type, placement, arrangement, construction, etc.),different mirrors and beam splitters (e.g., different in terms of type,location, angle, etc.). Thus, different embodiments can comprise, e.g.,λ/2 waveplates, λ/4 waveplates (e.g., when the input polarization iscircular), λ/n waveplates of other specific retardation, etc., and cancomprise at least 1 waveplate; at least 2 waveplates, at least 3waveplates, or at least 5 or more waveplates in various arrangements.The waveplates of the invention are not necessarily limited by theirconstruction. Thus, solid crystal (e.g., crystal quartz, or any otherappropriate substance) and liquid crystal waveplates are includedherein.

While the current embodiment includes, and is described throughout interms of its interaction with, nucleic acid sequencing systems (e.g.,various sequencing by synthesis configurations as described herein), itwill be appreciated by those of skill in the art that the currentinvention is also applicable to a wide range of other uses/systems.Thus, the embodiments can include systems comprising one or morewaveplate (typically rotating) that mixes the optic modes of a multimodeoptical fiber in order to produce a spatially substantially uniformimage or output in a desired timeframe (e.g. such as during the imagecapture time for a camera or the like). The current waveplate aspectscan optionally be utilized with systems, such as those trackingfluorescence on a plate or microarray or the like, that is not asequencing reaction. Correspondingly, the waveplate aspects can alsoinclude methods to create a substantially uniform image or output from amultimode optic fiber in a desired timeframe by passing the optic modesof the fiber through one or more waveplate (typically rotating andtypically rotating at a speed faster than the image capture time ordesired timeframe).

Various images obtained from exposure of a camera from such exemplaryembodiments are shown in FIG. 41. From a single exposure, the prominentregions of bright and dark pixels are evident in FIG. 41A. Thebright/dark images result from constructive and destructive interferenceof various modes that are present in the multimode optical fiber.Rotation of the waveplates results in spatial redistribution of the darkand bright regions as shown in the series of images in FIG. 41B. In suchFigures, each image was taken at a different waveplate setting. Asmentioned previously, if the waveplate is rotated faster than the imageacquisition time, then the spatial profile is averaged and uniformsmoothing of the image results. Such uniform smoothing is comparable toobtaining a large number of images and averaging them. FIGS. 41C and 41Dshow a single image with its line profile (41C) and an average of 54images with associated line profile (41D). FIG. 42, shows thesubstantial uniformity of the footprint produced by use of thewaveplate(s).

Other methods of ensuring that the optical beam is uniform over theimaging footprint include the use of solenoids, rotation of the lightbeam in an electric or magnetic field using Faraday or Pockel cells, andreimaging the light after it has gone through a diffuser. The diffusercan be a holographic diffuser that would superimpose light wavesoriginating at the end of the fiber (if fiber coupled) or at the laser(if no fiber were present) in such a way that the waves superimpose andproduce the required beam shape. One such example is a diffuser with anintensity profile of sine(x)^2 (sine is sin(x)/x) which will transform agauss beam into a top-hat beam.

The various mode scrambling aspects herein can optionally becontrolled/manipulated through the one or more computer component andare typically coordinated/synched with the light illumination and lightdetection components (also typically by the computer aspects herein).

Devices for Detecting Fluorescence

There are numerous devices for detecting fluorescence, for examplephotodiodes and cameras, that can comprise the detection/detectorcomponent(s) of the current invention. In some embodiments herein, thedetector component can comprise a 1 mega pixel CCD-based optical imagingsystem such as a 1024×1024 back thinned CCD camera with 8 μm pixels,which at 40× magnification can optionally image an area of 0.33×0.33 mmper tile using a laser spot size of 0.5×0.5 mm (e.g., a square spot, ora circle of 0.5 mm diameter, or an elliptical spot, etc.). The camerascan optionally have more or less than 1 million pixels, for example a 4mega pixel camera can be used. In many embodiments, it is desired thatthe readout rate of the camera should be as fast as possible, forexample the transfer rate can be 10 MHz or higher, for example 20 or 30MHz. More pixels generally mean that a larger area of surface, andtherefore more sequencing reactions, can be imaged simultaneously for asingle exposure. This has the advantage of requiring fewer stage movesand filter wheel changes, and helps to speed up imaging. In particularembodiments, the CCD camera/TIRF lasers herein are capable of collectingabout 6400 images to interrogate 1600 tiles (since images are optionallydone in 4 different colors with optionally different filters in place)per cycle. For a 1 Mega pixel CCD, certain images optionally can containbetween about 5,000 to 50,000 randomly spaced unique nucleic acidclusters (i.e., images upon the flowcell surface). The theoreticaldensity of resolvable clusters per unit area (or image) is dependant ofthe size of the clusters, as shown in FIG. 29 which shows a 1 Mpix imageof the number of detected clusters as a function of total cluster numberand minimum cluster area. At an imaging rate of 2 seconds per tile forthe four colors, and a density of 25000 clusters per tile, the systemsherein can optionally quantify about 45 million features per hour. At afaster imaging rate, and higher cluster density, the imaging rate can besignificantly improved. For example at the maximum readout rate of a 20MHz camera, and a resolved cluster every 20 pixels, the readout can be 1million clusters per second. The instrument can be configured to havemore than a single camera. The light can be split to simultaneouslyimage two colors onto two cameras, or even four colors onto fourcameras. If four cameras are used in parallel, it is thus possible tosequence 1 million bases per second, or 86.4 billion bases per day.

There are two ways of splitting up the optical signals for a two camerasystem. If two lasers are used, there may be a red excitation and agreen excitation, with half the emission light split towards eachcamera. Alternatively both lasers may be used in both illuminationcycles, and the light may pass through a suitable dichroic mirror, sosending the red light in one direction, and the green light in adifferent direction, as shown in FIG. 36. Such system prevents thesignal losses associated with beam splitting, but does mean that two ofthe dyes are exposed to the laser before their intensity is recorded. Insome such embodiments, the excitation blocker, e.g., as shown in FIG. 36can comprise a dual notch filter (e.g., 532 and 660 nm). A picture of aninstrument with two detection cameras 3700 and two fluidics systems 3701(and two flowcells 3702) is shown in FIG. 37.

A “tile” herein is functionally equivalent to the image size mapped ontothe substrate surface. Tiles can be, e.g., 0.33 mm², 0.5 mm², 1 mm², 2mm² etc, although the size or the tile will depend to a large extent onthe number and size of pixels on the camera and the desired level ofmagnification. Also, it will be appreciated that the tile does not haveto equal the same size or shape as the illumination footprint from thelaser (or other light source), although this can be advantageous if theminimization of photobleaching is desired.

As stated previously, in the various embodiments herein, thecamera/laser systems collect fluorescence from 4 different fluorescentdyes (i.e., one for each nucleotide base type added to the flowcell).Again, additional material on other aspects of, and other conceptsregarding, SBS sequencing can be found in applicants' co-pendingapplications, for example WO04018497, WO04018493 and U.S. Pat. No.7,057,026 (nucleotides), WO05024010 and WO06120433 (polymerases),WO05065814 (surface attachment techniques), and WO 9844151, WO06064199and WO07010251 (cluster preparation and sequencing).

FIGS. 1 and 13-16 show various possible configurations of the camerasand lasers of the present invention, including a backlight design, aTIRF Imaging configuration, a laser focusing configuration, awhite-light viewing configuration, and an alternative laser focusingdesign. The white light excitation source is optional, and can be Usedas well as, or instead of, the excitation lasers. FIG. 1 shows thebacklight design system whilst recording an image in the TIRF imagingconfiguration. The configuration in FIG. 1 for the TIRF imaging isoptionally a configuration of the backlight design set-up shown in FIG.13. In FIG. 1, one of the two lasers (in laser assembly 160) is used toilluminate the sample (in flowcell 110), and a single one of the fouremission filters (in filter switching assembly 145) is selected torecord a single emission wavelength and to cut out any stray laserlight. During imaging, both focus laser (150) and optional white lightlamp (165) do not illuminate the sample as they are either blocked witha shutter or switched off. Laser illumination 101 and illumination fromthe flowcell up through the lens objective and camera 102 are alsoshown. FIG. 13 shows all the components of the system in the backlightdesign but without the specific TIRF imaging configuration. Cf. FIGS. 1and 13. Thus FIG. 13 shows: fluid delivery module 1300, flowcell 1310,waste valve 1320, temperature actuator 1330, heating/cooling component(e.g., Peltier) 1335, camera (e.g., CCD camera) 1340, lens objective1342, filter switching assembly 1345, focusing laser assembly 1350,excitation lasers assembly 1360, low watt lamp 1365, precision XY stage1370, focus (z-axis) device 1375, mirror 1380, “reverse” dichroic 1385,and laser fiber optic 1390.

FIG. 14 shows a similar system as that in FIG. 1, but in the laserfocusing configuration where the excitation lasers (in laser assembly1460) and optional white light 1465 are switched off. Focusing laser1450 is on and shines into the system, hits beam splitter 1485 (e.g., apick-off mirror 1% beam splitter) which direct a faint beam 1402 downthe objective to hit a small spot on the sample (in flowcell 1410). Thescattered light from the sample returns up objective (1442) through anempty slot in filter wheel switching assembly 1445 and is imaged by CCDcamera 1440. The position of the spot on the camera is used to ensurethe sample is at the right distance from the objective, and thereforethe image will be in focus. The numbering of the elements in FIG. 14 issimilar to that of the elements in FIG. 13, but numbered as “14” ratherthan “13,” e.g., 1460 corresponds to a similar element as 1360, etc. Theautofocus system is described in more detail below.

FIG. 15 shows the optional white light viewing configuration, wherefocus laser 1550 and illumination lasers 1560 are off. In suchconfiguration the white light from low watt lamp 1565 goes into thesystem as beam 1503 and is imaged directly on the camera. Here too, thenumbering of elements, except for beam 1503, etc., follows that of FIGS.13 and 14. FIG. 16 shows an alternative focus configuration where thesystem contains second focusing camera 1641, which can be a quadrantdetector, PSD, or similar detector to measure the location of thescattered beam reflected from the surface. This configuration allows forfocus control concurrent with data collection. The focus laserwavelength is optionally longer than the reddest dye emission filter.

FIGS. 17-19 show various schematics for beam shape and dimensions forTIRF assays carried out with use of the current systems herein, whileFIG. 20 displays an optional embodiment of a TIRF prism for use with thesystems herein. Thus it will be appreciated that the shape (e.g., round,square, etc.) of the laser beams and/or of the imaging areas illuminatedby the laser beams can optionally vary between different embodiments.FIG. 17 shows the dimensions and geometries of the beam as it emergesfrom the fiber. In order to illuminate a circle on the substrate, thebeam must be projected from the fiber as an ellipse since the beam hitsthe substrate surface at an angle to the normal. Edge view 1700 ofcircle projected by ellipse 1730 (e.g., an elliptical beam shaperequired at fiber exit (looking down fiber centerline at 22°). The prismface partial outline 1710 is not to scale and the edge view 1720 ofellipse 1730 is shown on the minor axis. Likewise in order to illuminatea square on the substrate, the beam must be projected onto the surfaceas a rectangle, as shown in FIG. 18. In FIG. 18, rectangle 1830 isshown. A rec-elliptical beam shape is required at fiber exit (lookingdown fiber centerline at 22°). Edge view 1800 of the square projected byrectangle 1830 is also indicated as is prism face partial outline 1830(not to scale) and edge view 1820 of rec-ellipse 1830 (minor axis).

As shown in FIG. 19, the prism is designed to allow the imaging beam tohit the substrate surface at approximately 68° (relative to the normal)to achieve a total internal reflection and generate an evanescent beamthat excites the fluorophores on the surface. To control the path of thebeam through the prism, and therefore keep the illumination footprintdirectly over a stationary objective lens as the flowcell moves, theprism may have a geometry where the angle of the prism to the surface isalso 68°, thereby ensuring that the light always hits the prism at 90°.The desired geometry of the prism is more fully described in applicationWO03062897. and the exemplary size and geometry is shown in FIG. 20,etc.

Beam shape of the lasers herein is optionally controlled by polishingthe multimode fiber output end in order to create, e.g., a square beam.See, e.g., FIG. 21. FIG. 21 shows imaged beam results from suchpolishing. In other embodiments, the beam is optionally round. In someinstances, the beam properties may be a Gaussian profile with thefollowing properties: a nominal image size of radius 0.17 mm, a maximalspot size of 0.25 mm radius, and 0.32 mm as the point after which thereis effectively no laser intensity. In certain embodiments, the beamintensity is greater than 90% maximum intensity at all positions withinthe nominal image size; 80% maximum intensity at all positions withinthe maximal spot size, and no greater than 1% of maximal intensityoutside of 0.32 radius. In various embodiments (in the absence of adynamic mode scrambler), the intensity at any point does not vary bymore than 5% RMS within the timescale of 1 s-1 h and the variation inthe total (integrated) laser power is not more than 3% RMS measured over24 hours.

Illumination Systems

A variety of illumination systems may be used in devices according tothe present invention. The illumination systems can comprise lampsand/or lasers. The systems can contain one or more illumination lasersof different wavelengths. For example the systems herein may contain twolasers of 532 nm and 660 nm, although lasers with other wavelengths mayalso be used. Additionally, in various embodiments, the lasers in thesystems herein are actively temperature controlled to 0.1 C, have TTLmodulation for the 660 nm laser diode with rise time less than 100 ms;have integrated manual shutters for fast modulation of the 532 nm laser,have integrated beam shaping optics to ensure the optimum beam aspectratio is maintained at the instrument interface to maximize signal tonoise ratio, have integrated mode scrambler to reduce ripple on theoutput of the multi-mode fiber, and have minimal heat generation. Theshutters and TTL modulation are used to ensure that the illumination isonly on the sample surface whilst the camera is recording images.Illumination of fluorophores can cause photobleaching, and thereforeexposure of substrates to the laser when not needed is generallyminimized, especially before the images are recorded.

FIGS. 22A and B give various filter wheel arrangements of certainembodiments for use with various optic configurations. The use of a twolaser excitation system to detect four fluorophores means that two ofthe fluorophores are excited away from their maximum absorbtionwavelength, as shown in FIG. 22B. If the emission filters used in allfour channels were the same band width, then the two fluorophoresnearest the 532 and 660 nm lasers would be significantly brighter thanthe two fluorophores excited further from the lasers. However, thisfactor can be negated by changing the bandwidth of the filters. Forexample, as shown in FIG. 22B, in the case of the 532 nm laser, dyesthat absorb at for example 530 and 560 nm can both be excited by the 532laser. The use of a narrow filter, close to the laser, for example a560/20 that lets light through from 550-570 nm only allows the lightfrom the 532 nm dye through. The use of a wider bandpass filter furtheraway from the laser, for example a 610/75 that allows light through from572 nm to 647 nm lets through the light from both fluorophores. Theintensity of the 532 fluorophore through the 560/20 filter is similar tothe intensity of the 560 fluorophore through the 610/75 filter. Thus thetwo fluorophores can be clearly distinguished using a single laser andtwo emission filters.

The effect is not wavelength specific, and can be performed using anyexcitation wavelength. The same effect can therefore be achieved usingthe red laser. Two fluorophores that absorb at 650 nm and 680 nm can bedistinguished using a narrow filter close to the laser (for example a682/22), and a broader filter further away, for example a 700 long pass.Again the intensities of the two dyes through their respective filtersis similar, whilst the signal from the 680 dye in the 682/22 filter ismuch reduced. Both dyes emit into the 700 long pass channel, but thesignals can clearly be determined due to the different level of emissionin the narrow filter. The adaptation of laser wavelengths, fluorophoreselections and filter bandwidths can be used to obtain a set of fourfluorophores using any number of wavelengths, and the intensities of theemission through each channel can be normalized using the bandwidth ofthe filters to control how much light is transmitted.

FIG. 23 shows a nominal design for an embodiment of the 30× system raytrace, while FIG. 24 shows the 30× imaging performance. The imagingperformance of the system is dependent on the magnification of theobjective lens, and the other lenses in the system. A smallermagnification will allow a larger area of the substrate to be imaged,but at a cost of resolution of closely packed clusters and thebrightness of each cluster. A preferred magnification is optionallybetween 10×-40×, for example 20× or 30×. The objective can be customdesigned to allow diffraction limited imaging to be retained whenviewing fluorescence objects through a non-standard geometry (forexample thicker glass substrates) and hence removing the otherwisepresent spherical aberration. The objective lens may be connected to thedetector via a further tube lens.

Autofocus System

In particular embodiments, the systems herein can comprise components toaid in proper focusing of imaging clusters. In general, in particularembodiments herein, in an autofocus set up, an autofocus laser beamshines down to a sample through an objective lens, reflects from theflowcell surface, goes back to the lens and then to the camera, thuscreating a spot on the image. When the objective is moved up/down with afixed sample, the spot centroids align around a straight line on theimage (calibration curve). Displacement “dr” along this calibration lineis proportional to the change “d(z−zf)” in the distance between theobjective and the focal plane. In many embodiments, before the run, thesoftware establishes the orientation of the calibration line (its slope)and the “sensitivity”: dz/dr (nm/pixel). This is accomplished by taking21 images with the step of 1000 nm in z-direction around focus positionwhich is established visually. The software also can require the x, ypixel coordinates of the spot when the sample is in focus: x_(f), y_(f),this is determined from the first (central) focus image from the set ofthe 21 calibration images. For example, the devices herein optionallycomprise an auto focus function objective achieving 100 nm resolutionmounted with up to 50 mm Z axis motion. The objective lens canoptionally move vertically in relation to the substrate, and theillumination laser can be coupled to the Z axis motion such that theillumination inputs also move in relation to the substrate. Forembodiments having autofocus capability, an auto focus beam isoptionally sent along the edge of the microscope objective lens(optionally as far off-axis as possible in order to correspond tomaximum sensitivity). The autofocus beam can come from the illuminationlasers, or from a separate source that is optionally a differentwavelength than the illumination laser, for example 488 nm, 630 nm or aninfra-red laser of 700 nm or redder. The reflected beam is thenoptionally monitored by either a quad cell or by leakage through adichroic beam splitter onto the fluorescence imaging camera. In suchembodiments, the lens and camera are optionally the same as that used inthe instrument (e.g., 20× lens). Similar autofocus systems which areoptionally included within the current systems and devices have beenpreviously described, for example in WO03060589.

With particular autofocusing aspects herein, as the imaging plane moveswith respect to the objective lens, the reflected monitoring beam alsooptionally moves laterally (i.e. dotted line is the in focus plane whilethe solid line represents an out of focus plane which gives rise to alateral shift in the detected beam in FIG. 25). The dichroic mirrorchosen in such embodiments is usually one that reflects the autofocusbeam. The small leakage that is actually transmitted (c.a. <5%) is morethan adequate to observe on a CCD camera with no emission filter in theemission path. FIG. 26 shows sample photographs of both out of focus andin focus images where the spot is seen on the imaging camera. The lowerimages show the detected autofocus spot on the imaging camera. The spotcan also be seen on a separate detector, as shown in FIG. 27.

In embodiments comprising autofocus aspects, the computer componentoptionally comprises an autofocus algorithm. Such algorithms optionallyaid in the determination of the correct focus (e.g., by monitoring theabove measurements and adjusting accordingly). The autofocus spot can bemade to move in a 1D manner e.g. in just the y-direction rather than x &y, thus simplifying the procedure. The focus position of the objectivelens is assumed to move in the z-direction.

The first step in some embodiments of the autofocus analysis is a “SetupResponse function” wherein positions of the autofocus spot (y₁, y₂ . . .y_(n)) are measured on the imaging camera for several positions of theobjective lens (z₁,z₂ . . . z_(n)). Typically 5 positions are adequate.Shown in FIG. 27 is an analysis with just 3 positions. The movement ofthe reflected spot is shown as imaged on a fluorescence camera in thelower panels. For each Z plane there is an associated y-position of thereflected spot (centroid) on the camera. These five data points (z₁,y₁), (z₂, y₂), (z₃, y₃), (z₄, y₄), (z₅, y₅) can be described by a line.z=my ₀ +c   Equation 1The m and c values are given from the data points from least squaresfits as:

$\begin{matrix}{c = \frac{{\sum{z \cdot {\sum y^{2}}}} - {\sum{y \cdot {\sum({yz})}}}}{{5{\sum y^{2}}} - \left( {\sum y} \right)^{2}}} & {{Equation}\mspace{14mu} 2} \\{m = \frac{{5 \cdot {\sum({yz})}} - {\sum{y \cdot {\sum z}}}}{{5{\sum y^{2}}} - \left( {\sum y} \right)^{2}}} & {{Equation}\mspace{14mu} 3} \\{y_{0} = {- \frac{c}{m}}} & {{Equation}\mspace{14mu} 4}\end{matrix}$Thus, from the 5 data points, the values of c, m and y₀ are determinedgiving a known response function.

The next step in such embodiments of the autofocus analysis comprises“Calculate newC (for out of focus position)” wherein for each position cis constant, i.e. doesn't change for an out of focus or in focusposition. However, it does change for different positions. Hence as thestage moves to a new position NewC is calculated from the changed Z andy values as:newC=Z _(measured) −m.y _(measured)   Equation 5

The third step in the process is to use newC to calculate required Zposition (newZ) to get in focus y position (y₀). It is known thatnewC=newZ−m.y ₀   Equation 6newZ=newC+m.y ₀   Hence Equation 7m & y₀ are measured from step 1 (once per chip). newC is measured fromstep 2 (every position). Hence newZ can be calculated.

Another aspect in auto focus components of the invention comprises laserpointing stability requirements. To assess how much pointer error can betolerated in the auto focus laser, one can view the objective as asimple thin lens with the proper focal length as shown in theexaggerated drawing in FIG. 28.

Simple geometry y then yields that the angle Φ that would cause the autofocus laser beam to appear shifted by one pixel is simply at a (Δ/F)which is approximately Δ/F for small angles.

For a 20× objective (with the tube lens relay lens combination presentin some embodiments) the pixel size is roughly 0.3 μm. The focal lengthof that lens is 10 mm. Hence the error angle that corresponds to 1 pixelis approximately 30 μrad. Some embodiments of the system have their autofocus set for a sensitivity of about 4 pixel shift of the auto focuslaser spot per micron of z motion.

Assuming that 0.5 μm of focus error (corresponding to two pixels shiftin the position of the laser spot) can be tolerated, it is seen that thebiggest pointing change that can tolerate for the auto focus laser is 60μrad.

To meet that sort of stability requirement over normal room temperaturevariations, it is highly recommendable to use a fiber optically coupledlaser. Unless a solid state laser is very carefully temperaturecontrolled, it will be difficult to maintain this sort of pointingaccuracy within a reasonable ambient temperature spec range (e.g., 20-30C.).

To provide additional information and theory in regard to focus trackingalgorithms, a more detailed example of implementing the autofocus systemis given below.

The first step in some embodiments of the focus tracking procedure is toobtain the image of the autofocus laser spot on an imaging device, whichmay be the imaging camera. Data from this primary spot is extracted intwo passes—a first coarse pass that determines the approximate positionand size of the spot, followed by a second fine pass that determines thespot boundaries correctly before determining the COL (Center of Light)and other spot features. The first pass analysis can be performed in 5steps: (1) the 16-bit image is converted to an 8-bit image, with maximumof the image set to 255, minimum of image set to 0, and all othergrayscale values linearly in between; (2) The Picture Quality of theimage is computed. Picture quality is defined as the average of thenormalized autocorrelation of the image with itself with shifts of unitpixel to the left and unit pixel down. If the image is noisy, then sincenoise does not correlate with itself, this measure will be low; (3)Next, this image is thresholded at 128. Anything above this value willbe regarded as foreground, while anything below this level will be seenas background. Starting from the grayscale 255 (i.e. the hotspots),region-grow to find all 8-connected foreground components; (4) Of allthese candidate foreground components, the component with the highestaverage brightness is chosen as “the” component specifying the positionand approximate size of the primary spot; and (5) The bounding box ofthis component is computed.

The second (fine) pass analysis is performed in three steps: (1) Thesub-image corresponding to twice the area of the bounding box is cut outfrom the 8-bit grayscale image. This makes the population of foregroundand background pixels approximately equal, thereby making it easier forstandard image histogram based thresholding techniques to work reliably;(2) The histogram of the subimage is computed and the “best” grayscalethreshold that separates foreground from background is determined. Thethreshold used is called “Otsu's Threshold” (see IEEE Trans. Systems,Man, and Cybernetics, vol. 9, pp. 62-66, 1979, or Computer and RobotVision, volume 1. Addison-Wesley. 1992.); and (3) The image isthresholded at the Otsu threshold and the 8-connected foregroundcomponent is determined. This is the primary spot blob on which everysubsequent feature extraction is carried out.

An additional pass can be carried out to extract the position of thesecondary spot. Four steps can be done to carry out such additionalpass: (1) The 8-bit image (from the First Pass) is thresholded at a lowthreshold of 16; (2) It should be noted that at this lower threshold,the number of pixels (area) of the primary spot component increases.This area of the primary spot is recorded and is used to determine howtight or diffuse it is; (3) The component (of sufficient size) closestin distance to the primary spot component is identified as the secondaryspot; (4) The geometric centroid of the secondary spot is recorded.

Center of Light Determination

To determine the Center of Light (COL) for autofocusing, ( x, y) isdenoted as the center of light of the primary spot. Then this iscomputed for the primary spot blob as:

${\overset{\_}{x} = \frac{\sum{g_{i}x_{i}}}{\sum g_{i}}}\mspace{14mu}$and ${\overset{\_}{y} = \frac{\sum{g_{i}y_{i}}}{\sum g_{i}}},$where the summation is taken over all pixels i in the blob having imagebased coordinates (x_(i),y_(i)) and grayscales g_(i) (above threshold).

Other Spot Features

In addition to Picture Quality and the Center of Light, the list offeatures calculated for the primary spot blob includes Area which is ameasure of how diffuse (non-tight) the primary blob is. This is set tothe area (count in pixels) of the primary blob at the low thresholddivided by its area at the Otsu threshold. Other features calculatedinclude Volume (the average brightness, above threshold×Area of theprimary blob); Average Brightness, which is the sum of the gray valuesof the pixels in the blob divided by its area; and Maximum Brightness:Maximum gray value.

The extracted data can then be used to calibrate the Z focus, andthereby determine how much, to move the objective in order for the imageto be in focus. The overview of the calibration procedure is as follows:(1) Calibration for the Z focus is done (with user help) at thebeginning of every run; (2) At the beginning of calibration the usermakes sure that the image is in focus, i.e. at the focal plane. He/shethus sets the Z focus point z_(F); (3) This embodiment of autofocusrelies on user input and the coordinates of the autofocus laser spot onan image; (4) As z is changed during the Calibration process, the spotmoves in linear proportion to the change of z along a line.

The calibration algorithmic procedure begins with a sequence of Centerof Lights (x, y) extracted from the sequence of autofocus spot imagesacquired as z is changed. Ideally these points when graphed should allfall perfectly on a straight line, shown below.

Unfortunately, because of various noise sources, both physical andcomputational, the points are displaced from the ideal straight line, asshown:

Therefore an XY Principal Component Analysis (see e.g. I. T. Jolliffe,Principal Component Analysis, 2nd ed. Springer Series in Statistics,2002.) based regression is performed between X and Y, leading to a newcoordinates system R and Q as shown:

It should be noted that: (1) Origin of the (R, Q) system is at thecenter of mass of the (x, y) points; (2) A best fitting (principalcomponent) line defines the R axis; and (3) The orthogonal line to the Raxis defines the Q axis. The model calls for high correlation between Xand Y. The Q coordinate values can therefore be regarded as error“residuals” from the best fitting line—the idea is to get rid of theseresiduals and correct the observations.

Finally, since the model calls for a linear relationship between thespatial coordinates and the z values (as shown), a linear regression isperformed between r and z to determine the coefficients of this line:

Additionally autofocus tracking can involve various training ofregression engines.

Training the XY PCA Regression Engine

Input: A sequence of Center of Lights (x, y) extracted from the sequenceof autofocus spot images acquired as z is changed. Output: The PCARegression Coefficients, i.e. transformation to the (R, Q) coordinatespace. Description: Perform Principal Component Analysis and save thecoefficients. Also, given the centroid position of the secondary spot,determine whether it is to the left or the right of the primary spotalong the principal axis.Training the RZ Linear Regression Engine

Input: The r coordinates obtained from the PCA Regression, thecorresponding z values, and the Z focus point z_(F). Output: The LinearRegression Coefficients relating (z − z_(F)) to r. Description: PerformLinear Regression Analysis and save the coefficients.Training the Outlier Detector

An outlier detection scheme is used to warn the presence of a bubble orto flag a filter wheel problem.

Input: The q coordinates obtained from the PCA Regression, along withthe spot features for every spot in the calibration sequence are used totrain a classifier to detect outlier spots. Output: Internal settings ofthe Outlier Detector. Description: For every feature φ, the mean μ_(φ)and standard deviation σ_(φ) is calculated from the spots used duringcalibration. The general idea is that a spot would be declared anoutlier spot if the feature φ is outside the bounds of μ_(φ) ± 3σ_(φ).However in the current implementation only the Volume and Area featuresare used in this way—in fact, only the upper bounds are currently usedfor these two features. Special bounds are set to override these valuesfor both the picture quality and the q residue feature. For the picturequality, a lower bound is used. For the residue, an upper bound is used.Running the Autofocus System

A run starts from an image of the autofocus laser spot and uses thecoefficients of the transformations learned during the calibrationprocess to make the best estimate for the z displacement required tomove to focus.

Input: An image of the autofocus laser spot. Output: (z − z_(F)) to moveto focus. Also provides recommendation of whether or not to move basedupon outlier detection. Description: Derives best estimate of (z −z_(F)) using: 1. The spot extraction algorithm. 2. The PCA based XYRegression coefficients. 3. The Linear RZ Regression coefficients. Inone implementation, the outlier detection scheme allows a move only inthe case of a large q residual. In case of low picture quality, highvolume, or high area, it recommends non-movement. If during long periodsof z non-movement, as may be triggered by large bubbles/contaminationsin the flowcell. the surface of the flowcell drifts enough to make thesecondary spot appear locally in all respects to the primary spot seenin calibration, such algorithm would latch onto such spot for the restof the cycle even though the bubble/contamination ceased to exist insidethe flowcell. In order to recover from such problem, the following stepscan be taken: In case a move is recommended, a further check can beperformed to see whether a secondary spot exists in the directionopposite to the one where training says it ought to be. If it does, thena move can be recommended to this sport using the PCA based XYRegression and the Linear RZ Regression coefficients.

Computer

As noted above, the various components of the present system are coupledto an appropriately programmed processor or computer that functions toinstruct the operation of these instruments in accordance withpreprogrammed or user input instructions, receive data and informationfrom these instruments, and interpret, manipulate and report thisinformation to the user. As such, the computer is typicallyappropriately coupled to these instruments/components (e.g., includingan analog to digital or digital to analog converter as needed).

The computer optionally includes appropriate software for receiving userinstructions, either in the form of user input into set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations(e.g., auto focusing, SBS sequencing, etc.). The software then convertsthese instructions to appropriate language for instructing the correctoperation to carry out the desired operation (e.g., of fluid directionand transport, autofocusing, etc.).

For example, the computer is optionally used to direct a fluid flowcomponent to control fluid flow, e.g., through a variety of tubing. Thefluid flow component optionally directs the movement of the appropriatebuffers, nucleotides, enzymes, etc., into and through the flowcell.

The computer also optionally receives the data from the one or moresensors/detectors included within the system, and interprets the data,either provides it in a user understood format, or uses that data toinitiate further controller instructions, in accordance with theprogramming, e.g., such as in monitoring and control of flow rates,temperatures, and the like.

In the present invention, the computer typically includes software forthe monitoring and control of materials in the flowcells. Additionallythe software is optionally used to control excitation of the fluorescentlabels and monitoring of the resulting emissions. The computer alsotypically provides instructions, e.g., to the heating/cooling componentand autofocus system, etc.

Any controller or computer optionally includes a monitor, which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display), or the like.Data produced from the current systems. e.g., nucleic acid sequenceresults is optionally displayed in electronic form on the monitor.Additionally, the data, e.g., light emission profiles from the nucleicacid arrays, or other data, gathered from the system can be outputted inprinted form. The data, whether in printed form or electronic form(e.g., as displayed on a monitor), can be in various or multipleformats, e.g., curves, histograms, numeric series, tables, graphs andthe like.

Computer circuitry is often placed in a box which includes, e.g.,numerous integrated circuit chips, such as a microprocessor, memory,interface circuits. The box also optionally includes a hard disk drive,a floppy disk drive, a high capacity removable drive such as a writeableCD-ROM, and other common peripheral elements. Inputting devices such asa keyboard or mouse optionally provide for input from a user and foruser selection of sequences to be compared or otherwise manipulated inthe relevant computer system.

Exemplary Use and Component Variation

The SBS systems herein, in many embodiments, comprise CCD/TIRF laserbased excitation and imaging subsystems which can image millions ofnucleic acid clusters per sample (typically within a flowcell) and whichcan detect each of four fluorescent dyes (one for each of the fourbases). The SBS chemistry components, e.g., nucleotides. WO04018497,WO04018493 and U.S. Pat. No. 7,057,026, polymerases WO05024010 andWO06120433, surface attachment techniques. WO05065814, clusterpreparation and sequencing, WO 9844151, WO06064199 and WO07010251, arecompatible with the channeled flowcell components herein, etc. Thecomputer or data analysis system aspects of the system are optionallycapable of processing thousands of images per hour into sequenceinformation

As an overview, in particular examples of sequencing by SBS, genomic DNAis randomly fragmented, end capped with known sequences, and covalentlyattached to a substrate (such as the channel in a flowcell), e.g., byhybridization to a covalent primer. From such attached DNA, an array ofnucleic acid clusters is created, as described in WO9844141 andWO07010251. SBS analysis (e.g., using the systems and devices herein)can generate a series of images of the clusters, which can then beprocessed to read the sequence of the nucleic acids in each clusterwhich can then be aligned against a reference sequence to determinesequence differences, a larger overall sequence, or the like. Algorithmsfor the alignment of short reads of nucleic acids are described inWO05068089.

As described above, each sequencing cycle will include a round ofincorporation onto the growing nucleic acid chain. Such cycle istypically done by an addition of all four dNTPs, each modified so thateach base is identifiable by a unique fluorophore. Additionally, thetriphosphates are modified at the 3′ position so that extension iscontrolled and not more than a single base can be added to each moleculein each cycle. The generic concept of performing clusters amplified froma single template molecule on a random array, and the subsequentsequencing of said array is shown in FIGS. 30-32, which schematicallyillustrate various aspects of sequencing procedures and methods carriedout by the systems herein. For example, the basic overview steps offormation of nucleic acid clusters, the cluster arrays produced (and acomparison of such cluster arrays against a more “traditional” array)and an outline of the sequencing methodology are all presented. FIG. 30shows the basic outlines of nucleic acid cluster creation and sequencingwhile FIG. 31 compares nucleic acid density between an array (on left)and on a nucleic acid cluster substrate such as those capable of usewith the systems/devices of the invention (on right). FIG. 32 gives acartoon outlining the SBS sequencing procedure, e.g., as done byembodiments of the invention.

After the incorporation step wherein a fluorescently labeled nucleotideis bound to the nucleic acid of the members of the clusters through acleavable linker, the channels of the flowcell are washed out by thefluid flow subsystem in order to remove any unincorporated nucleosidesand enzyme.

Next, a read step is performed by the system, whereby the identity ofthe individual labels (read as a group in each cluster) incorporated inthe incorporation step is recorded using optical microscopy and thecorresponding base incorporated is noted. The sequencing system can readthe four different fluorophores using two lasers at distinct wavelengthsvia total internal reflection microscopy (TIRF) and four distinctemission filters at different parts of the spectrum. The images arerecorded onto a CCD camera and reported into the attached computermodule.

After the specific incorporation is read, a deprotection step removesthe labeling moiety and block from the surface bound DNA. Thedeprotection allows repetition of the above incorporation and readingsteps until sufficient cycles of information are obtained to uniquelyplace the sequence of each nucleic acid cluster (present on theflowcell) in its genomic context. For example, in the case of the humangenome this will be >16 cycles, e.g., about 25-50 cycles. The images canbe stored off line, or processed in real time such that the individualbases are read during the sequencing process. Processing the imagesprovides a database of a sequence read from every cluster, where eachcluster is derived from a random position somewhere in entire sample(e.g., a genome). Thus during the course of the procedure, a database ofmillions of sequence reads covering every part of the genome istypically constructed. Such database can be, e.g., compared with adatabase of every sequence derived from a reference sequence, etc. Invarious embodiments, image analysis, sequence determination, and/orsequence alignment are optionally performed “off-line” after thefluorescent images are captured. Such procedures are also optionallyperformed by a computer separate from the one present in the currentsystems.

As mentioned throughout, the current invention can vary betweenembodiments (e.g., in number and type of components or subsystems). Forexample, in one embodiment of the invention (embodiment “b”), thecomponents can comprise: illumination lasers (used to excite thefluorophores in the sequencing reactions) of 532 and 660 nm each with 75mW power (or optionally greater) that project as 0.5 mm circle on thebottom of the channel in a flowcell; a TIR prism of glass (68 deg or 71deg); a glass flowcell with channels of 1×61 mm area having 8 channelsthat are 100 μm deep (39×1 mm usable or accessible for viewing); anobjective lens in the camera component comprising a Nikon Plan Apo 20×,0.75NA (corrected for glass thickness); emission filters comprisingBandpass filters of 557±11 nm, 615±40 nm. 684±11 nm, and 740±50 nm (oroptionally filters as shown, or similar to those shown, in FIG. 22);relay optics comprising a Navitar 1.33× adapter for a net magnificationof about 23×, or an unmagnified tube lens; and a digital CCD cameracomprising a Photometrics Cascade 1 Mpix or 1K camera, with a pixel sizeof 8 μm, and a readout rate of 10 MHz, and a microscope objective of 20×magnification with 0.75 NA (numerical aperture). The Cascade embodiment“b” can give a net performance of 0.35 mm field with approximately 0.8μm optical resolution (somewhat larger than diffraction limit).

Such 1 Megapixel embodiments can illuminate a 0.5 mm circle and detect a0.35 mm square inside it. The flowcell in such embodiments can have atotal of 156 non-overlapping tiles in a channel or higher. The clusterscan be on the order of 1 μm. The NA of the microscope can optionallygive a PSF of approximately 0.6 μm at 700 nm. Thus, a “typical” clustergets an apparent diameter of approximately 1.2 μm. In the image plane, 1pixel represents approximately 0.35 μm, so a typical cluster would haveabout 3.5 pixels diameter. The area of a cluster is the about 9.25pixels. Poisson distribution of 10 area pixel objects on 1 Mpixel CCDshows maximum of about 38,000 objects will be non-overlapping as shownin FIG. 29. FIG. 29 gives an example of information throughput from anexemplary configuration of a system of the invention. The number ofdetected clusters is a function of the total cluster number and theminimum cluster area.

For exemplary “b” embodiments, the resolution limit (using Rayleighcriterion) is about 0.6 μm and clusters are about 1 μm for an apparentsize of about 1.2 μm. Pixels map to about 0.35 μm in image plane so acluster is about 3.5 pixels across and about 10 pixels in area. Forrandomly distributed clusters, the maximum number of unconfused clustersin the 1 MPix camera will be about 38,000 in a 0.35 mm square tile. “b”flowcells accommodate 150 non-overlapping illumination tiles per channelfor a total of 1200 tiles per flowcell. This is 45.6 M Bases per cycleand about 1 GBase in a 25 cycle run. Overlapping the illumination andclosely packing the tiles means that 200 tiles can be imaged perchannel, and therefore 1600 per flowcell.

For the “b” illumination subsystem throughput, the laser wavelengthsare: green laser wavelength 532 nm; green laser power optionally 75 mW;red laser wavelength 660 nm; red laser power optionally 75 mW; projectedTIRF beam diameter 0.5 mm; and allowed variation across beam 20%.

In another embodiment, (embodiment “g”), the system of the invention cancomprise: illumination lasers of 532 and 660 nm, each with 500 mW power(ideally projected as 0.5 mm square), a TIR prism of glass (68 deg); aglass flowcell having 8 channels 100 μm deep and of 1×61 mm in area witha 50 mm usable; an objective lens comprising a Nikon Plan Fluor 40×, 0.6NA adjustable collar, or custom 40×, 0.75 NA corrected for an SBSflowcell; emission filters comprising Bandpass filters of 557±11 nm,615±40 nm, 684±11 nm, and 740±50 nm; image optics comprising an 150 mmachromatic doublet for system magnification of 30×; and a digital CCDcamera comprising a Photometrics CoolSNAP K4, 2048 by 2048 pixels, 4Mpix camera, 7.4 μm pixel size, 20 MHz readout. Such embodiment can givea net performance of 0.5 mm field with less than 0.7 μm diffractionlimit. It can comprise a relay lens of 0.75× for total 30× systemmagnification.

In some such “g” embodiments, it is desired that a 0.5 mm square isuniformly illuminated and that the same 0.5 mm square is detected(2048×7.4/30000). The clusters on the flowcells herein can be as smallas 0.5 μm. PSF at 700 nm is approximately 0.7 μm. Clusters thus appearas 0.86 μm where 1 pixel represents 0.25 μm. A typical cluster thereforeis 3.5 pixels and the area of a cluster is 9.25 pixels. 4 Mpixel CCDgives a maximum of about 135,000 detectable non-overlapping clusters pertile.

The illumination footprint is four times larger, meaning a 4 timeincrease in laser powers is needed to obtain the same level of signal inthe same exposure time. To minimize exposure times, the laser power canbe increased further. Such a system is therefore capable of generating 2billion bases of sequence per experiment, if the following parametersare used: Objective with numerical aperture 0.8; 20× magnification; 4Mpixel camera; 760 μm×760 μm illumination tiles; 1 imaging lane per flowchannel; 48 tiles per lane; 8 channels per chip; clusters of averagesize 0.7 μm; and, read length of 40 bases. Therefore total throughput=8channels×48 tiles×135000 clusters/tile×40 cycles=2.07 billion bases (G).

Increasing the size of the flowcell to increase the numbers of tilesimaged, the density of clusters, or the read length, will enableimprovements in the number of bases generated per flowcell. Two or fourcameras can be mounted in parallel to obtain a system with two or fourtimes the throughput. A two camera configuration is shown in FIGS. 36and 37. The scanning time can be decreased using techniques such as TimeDelay Integration (TDI), meaning that the surface is continually scannedrather than imaged in discrete tiles. The instrument can be configuredto perform multiple chemistry steps with multiple fluidics systemscoupled to a single optical system. In the single chemistry system, theoptical system is not imaging whilst the chemistry steps are occurring.If the chemistry and imaging parts of the cycle take similar lengths,then for 50% of the time, the instrument is not recording data. If thescanning part of the system is further speeded up, then an even higherpercentage of the experimental run time is spent performing chemistry.This can be alleviated if the system is configured such that multipleflowcells are processed simultaneously, with one flowcell alwaysundergoing imaging. Schematic representations of a dual flowcell holderare shown in FIG. 43.

Although the system as described is shown with the illumination fromunderneath, and the objective on top, the system as shown can beinverted to illuminate from the top, and have the detection systemunderneath. See above. The heating and illumination can be carried outfrom either face of the substrate, so that bottom side heating and topside illumination are also within the scope of the invention. Theoperation of systems within the scope of the inventions are furtherdescribed in the following general methods.

Examples of Using the System in Sequencing

The following are examples of general techniques and the like (e.g., fornucleic acid cluster formation) which can optionally be applied in usewith the systems of the invention. It will be appreciated that suchdescriptions and examples are not necessarily limiting upon the currentsystems and their use unless specifically stated to be so. The methodsfor forming and sequencing nucleic acid clusters are fully described inpatent application WO07010251, the protocols of which are incorporatedherein by reference in their entirety, but some elements of theseprotocols are summarized below.

Preparation of Substrates and Formation of Nucleic Acid Clusters

Acrylamide Coating of Glass Chips

The solid supports used for attachment of nucleic acid to be sequencedare optionally 8-channel glass chips such as those provided by SilexMicrosystems (Sweden). However, the experimental conditions andprocedures are readily applicable to other solid supports as well. Insome embodiments chips were washed as follows: neat Decon for 30 min,milliQ H2O for 30 min, NaOH 1N for 15 min, milliQ H2O for 30 min, HCl0.1N for 15 min, milliQ H2O for 30 min. The Polymer solution preparationentailed:

For 10 ml of 2% polymerization mix.

-   -   10 ml of 2% solution of acrylamide in milliQ H2O;    -   165 μl of a 100 mg/ml N-(5-bromoacetamidylpentyl)acrylamide        (BRAPA) solution in DMF (23.5 mg in 235 μl DMF);    -   11.5 μl of TEMED; and,    -   100 μl of a 50 mg/ml solution of potassium persulfate in milliQ        H2O (20 mg in 400 μl H2O).

In such embodiments, the 10 ml solution of acrylamide was first degassedwith argon for 15 min. The solutions of BRAPA, TEMED and potassiumpersulfate were successively added to the acrylamide solution. Themixture was then quickly vortexed and immediately used. Polymerizationwas then carried out for 1 h 30 at RT. Afterwards the channels werewashed with milliQ H2O for 30 min and filled with 0.1 M potassiumphosphate buffer for storage until required.

Synthesis of N-(5-bromoacetamidylpentyl)acrylamide (BRAPA)

N-Boc-1,5-diaminopentane toluene sulfonic acid was obtained fromNovabiochem. The bromoacetyl chloride and acryloyl chloride wereobtained from Fluka. All other reagents were Aldrich products.

To a stirred suspension of N-Boc-1,5-diaminopentane toluene sulfonicacid (5.2 g, 13.88 mmol) and triethylamine (4.83 ml, 2.5 eq) in THF (120ml) at 0° C. was added acryloyl chloride (1.13 ml. 1 eq) through apressure equalized dropping funnel over a one hour period. The reactionmixture was then stirred at room temperature and the progress of thereaction checked by TLC (petroleum ether:ethyl acetate 1:1). After twohours, the salts formed during the reaction were filtered off and thefiltrate evaporated to dryness. The residue was purified by flashchromatography (neat petroleum ether followed by a gradient of ethylacetate up to 60%) to yield 2.56 g (9.98 mmol, 71%) of product 2 as abeige solid. 1H NMR (400 MHz, d6-DMSO): 1.20-1.22 (m, 2H, CH2),1.29-1.43 (m, 13H, tBu, 2×CH2), 2.86 (q, 2H, J=6.8 Hz and 12.9 Hz, CH2),3.07 (q, 2H, J=6.8 Hz and 12.9 Hz, CH2), 5.53 (dd, 1H, J=2.3 Hz and 10.1Hz, CH), 6.05 (dd, 1H, J=2.3 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 Hz, CH), 6.77 (t, 1H, J=5.3 Hz, NH), 8.04 (bs, 1H, NH). Mass(electrospray+) calculated for C13H24N2O3 256, found 279 (256+Na+).

Product 2 (2.56 g, 10 mmol) was dissolved in trifluoroaceticacid:dichloromethane (1:9, 100 ml) and stirred at room temperature. Theprogress of the reaction was monitored by TLC (dichloromethane:methanol9:1). On completion, the reaction mixture was evaporated to dryness, theresidue co-evaporated three times with toluene and then purified byflash chromatography (neat dichloromethane followed by a gradient ofmethanol up to 20%). Product 3 was obtained as a white powder (2.43 g, 9mmol, 90%). 1H NMR (400 MHz, D2O): 1.29-1.40 (m, 2H, CH2), 1.52 (quint.,2H, J=7.1 Hz, CH2), 1.61 (quint., 2H, J=7.7 Hz, CH2), 2.92 (t, 2H, J=7.6Hz, CH2), 3.21 (t, 2H, J=6.8 Hz, CH2), 5.68 (dd, 1H, J=1.5 Hz and 10.1Hz, CH), 6.10 (dd, 1H, J=1.5 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 Hz, CH). Mass (electrospray+) calculated for C8H16N2O 156,found 179 (156+Na+).

To a suspension of product 3 (6.12 g, 22.64 mmol) and triethylamine(6.94 ml, 2.2 eq) in THF (120 ml) was added bromoacetyl chloride (2.07ml, 1.1 eq), through a pressure equalized dropping funnel, over a onehour period and at −60° C. (cardice and isopropanol bath in a dewar).The reaction mixture was then stirred at mom temperature overnight andthe completion of the reaction was checked by TLC(dichloromethane:methanol 9:1) the following day. The salts formedduring the reaction were filtered off and the reaction mixtureevaporated to dryness. The residue was purified by chromatography (neatdichloromethane followed by a gradient of methanol up to 5%). 3.2 g(11.55 mmol, 51%) of the product 1 (BRAPA) were obtained as a whitepowder. A further recrystallization performed in petroleum ether:ethylacetate gave 3 g of the product 1. 1H NMR (400 MHz, d6-DMSO): 1.21-1.30(m, 2H, CH2), 1.34-1.48 (m, 4H, 2×CH2), 3.02-3.12 (m, 4H, 2×CH2), 3.81(s, 2H, CH2), 5.56 (d, 1H, J=9.85 Hz, CH), 6.07 (d, 1H, J=16.9 Hz, CH),6.20 (dd, 1H, J=10.1 Hz and 16.9 Hz, CH), 8.07 (bs, 1H, NH), 8.27 (bs,1H, NH). Mass (electrospray+) calculated for C10H17BrN2O2 276 or 278,found 279 (278+H+), 299 (276+Na+).

The Cluster Formation Process

Fluidics

For all fluidic steps during the cluster formation process, aperistaltic pump Ismatec IPC equipped with tubing Ismatec Ref 070534-051(orange/yellow, 0.51 mm internal diameter) is optionally used. The pumpis run in the forward direction (pulling fluids). A waste dish isinstalled to collect used solution at the outlet of the peristaltic pumptubing. During each step of the process, the different solutions usedare dispensed into 8 tube microtube strips, using 1 tube per chip inlettubing, in order to monitor the correct pumping of the solutions in eachchannel. The volume required per channel is specified for each step.

Thermal Control

To enable incubation at different temperatures during the clusterformation process, the Silex chip is mounted on top of an MJ-Researchthermocycler. The chip sits on top of a custom made copper block, whichis attached to the flat heating block of the thermocycler. The chip iscovered with a small Perspex block and is held in place by adhesivetape. Both pump and thermocycler are controlled by computer run scripts,which prompt the user to change solutions between each step.

Grafting Primers Onto Surface of SFA Coated Chip

An SFA coated chip is placed onto a modified MJ-Research thermocyclerand attached to a peristaltic pump as described above. Grafting mixconsisting of 0.5 μM of a forward primer and 0.5 μM of a reverse primerin 10 mM phosphate buffer (pH 7.0) is pumped into the channels of thechip at a flow rate of 60 μl/min for 75 s at 20° C. The thermocycler isthen heated up to 51.6° C., and the chip is incubated at thistemperature for 1 hour. During this time, the grafting mix undergoes 18cycles of pumping: grafting mix is pumped in at 15 μl/min for 20 s, thenthe solution is pumped back and forth (5 s forward at 15 μl/min, then 5s backward at 15 μl/min) for 180 s. After 18 cycles of pumping, the chipis washed by pumping in 5×SSC/5 mM EDTA at 15 μl/min for 300 s at 51.6°C. The thermocycler is then cooled to 20° C.

Template DNA Hybridization

The DNA templates to be hybridized to the grafted chip are diluted tothe required concentration (currently 0.5-2 pM) in 5×SSC/0.1% Tween. Thediluted DNA is heated on a heating block at 100° C. for 5 min todenature the double stranded DNA into single strands suitable forhybridization. The DNA is then immediately snap-chilled in an ice/waterbath for 3 min. The tubes containing the DNA are briefly spun in acentrifuge to collect any condensation, and then transferred to apre-chilled 8-tube strip and used immediately.

The grafted chip from above is primed by pumping in 5×SSC/0.1% Tween at60 μl/min for 75 s at 20° C. The thermocycler is then heated to 98.5°C., and the denatured DNA is pumped in at 15 μl/min for 300 s. Anadditional pump at 100 μl/min for 10 s is carried out to flush throughbubbles formed by the heating of the hybridization mix. The temperatureis then held at 98.5° C. for 30 s, before being cooled slowly to 40.2°C. over 19.5 min. The chip is then washed by pumping in 0.3×SSC/0.1%Tween at 15 μl/min for 300 s at 40.2° C. The script then runs straightto the next step.

Amplification

The hybridized template molecules are amplified by a bridging polymerasechain reaction using the grafted primers and a thermostable polymerase.Amplification buffer consisting of 10 mM Tris (pH 9.0), 50 mM KCl, 1.5mM MgCl2, 1 M betaine and 1.3% DMSO is pumped into the chip at 15 μl/minfor 200 s at 40.2° C. Then amplification mix of the above buffersupplemented with 200 μM dNTPs and 25 U/ml Taq polymerase is pumped inat 60 μl/min for 75 s at 40.2° C. The thermocycler is then heated to 74°C. and held at this temperature for 90 s. This step enables extension ofthe surface bound primers to which the DNA template strands arehybridized. The thermocycler then carries out 50 cycles of amplificationby heating to 98.5° C. for 45 s (denaturation of bridged strands), 58°C. for 90 s (annealing of strands to surface primers) and 74° C. for 90s (primer extension). At the end of each incubation at 98.5° C., freshPCR mix is pumped into the channels of the chip at 15 μl/min for 10 s.As well as providing fresh reagents for each cycle of the PCR, this stepalso removes DNA strands and primers which have become detached from thesurface and which could lead to contamination between clusters. At theend of thermocycling, the chip is cooled to 20° C. The chip is thenwashed by pumping in 0.3×SSC/0.1% Tween at 15 μl/min for 300 s at 74° C.The thermocycler is then cooled to 20° C.

Linearization

Linearization mix consisting of 0.1 M sodium periodate and 0.1 Methanolamine is pumped into the chip at 15 μl/min for 1 hr at 20° C. Thechip is then washed by pumping in water at 15 μl/min for 300 s at 20° C.

Blocking (Optional)

This step uses Terminal Transferase to incorporate a dideoxynucleotideonto the free 3′ OH ends of DNA strands (both grafted primers andamplified cluster molecules).

Blocking buffer consisting of 50 mM potassium acetate, 20 mMTris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol (pH 7.9) and250 μM CoCl2 is pumped into the chip at 15 μl/min for 200 s at 20° C.Then Blocking Mix of the above buffer supplemented with 2.4 μM ddNTPsand 250 U/ml Terminal transferase is pumped in at 15 μl/min for 300 s at37.7° C. The thermocycler is held at 37.7° C. for 30 min, during whichtime Blocking Mix is pumped into the chip at 15 μl/min for 20 s every 3min. After blocking, the chip is then washed by pumping in 0.3×SSC/0.1%Tween at 15 μl/min for 300 s at 20° C.

Denaturation of Clusters and Hybridization of Sequencing Primer

This step uses NaOH to denature and wash away one of the strands of theamplified, linearized and blocked clusters. After a wash to remove theNaOH, the sequencing primer is then hybridized onto the single strandsleft on the surface.

After blocking, the double stranded DNA in the clusters is denatured bypumping in 0.1N NaOH at 15 μl/min for 300 s at 20° C. The chip is thenwashed by pumping in TE (10 mM Tris pH 8.0, 1 mM EDTA) at 15 μl/min for300 s at 20° C. Sequencing primer is diluted to 0.5 μM in 5×SSC/0.1%Tween, and pumped into the channels at 15 μl/min for 300 s at 20° C. Thethermocycler is then heated up to 60° C. and held at this temperaturefor 15 min. The thermocycler is then cooled to 40.2° C. and the chip iswashed by pumping in 0.3×SSC/0.1% Tween at 15 μl/min for 300 s.

The clusters are now ready for 1st cycle sequencing enzymology, e.g.,with the systems and devices of the current invention.

The DNA sequence used in this process was a single monotemplate sequenceof 400 bases, with ends complimentary to the grafted primers. The duplexDNA was denatured as described above.

Grafting of Primers

The primers are typically 5′-phosphomthioate oligonucleotidesincorporating any specific sequences or modifications required forcleavage. Their sequences and suppliers vary according to the experimentthey are to be used for, and in this case were complementary to the5′-ends of the template duplex.

Sequencing of Linearized Clusters

The amplified clusters contained a diol linkage in one of the graftedprimers. Diol linkages can be introduced by including a suitable linkageinto one of the primers used for solid-phase amplification.

Suitable primers including any desired template-specific sequence can bemanufactured by standard automated DNA synthesis techniques usingcomponents available from commercial suppliers (e.g. Fidelity SystemsInc., ATD).

A cleavable diol-containing primer would typically have the followingstructure:

-   5′-phosphorothioate-arm 26-diol22A-sequence-3′OH.    Wherein “sequence” represents a sequence of nucleotides capable of    hybridizing to the template to be amplified.

The structures of the arm26 and diol22A components (from FidelitySystems Inc, MD, USA) are as follows:

Products containing such diol linkages can be cleaved using periodate asdescribed above, and the resulting single stranded polynucleotideshybridized as described above.DNA Sequencing Cycles

Sequencing was carried out using modified nucleotides prepared asdescribed in International patent application WO 2004/018493, andlabeled with four different commercially available fluorophores(Molecular Probes. Inc.).

A mutant 9°N polymerase enzyme (an exo-variant including the triplemutation L408Y/Y409A/P410V and C223S) was used for the nucleotideincorporation steps.

Incorporation mix, Incorporation buffer (50 mM Tris-HCl pH 8.0, 6 mMMgSO4, 1 mM EDTA, 0.05% (v/v) Tween-20, 50 mM NaCl) plus 110 nM YAVexo-C223S, and 1 μM each of the four labeled modified nucleotides, wasapplied to the clustered templates, and heated to 45° C.

Templates were maintained at 45° C. for 30 min, cooled to 20° C. andwashed with Incorporation buffer, then with 5×SSC/0.05% Tween 20.Templates were then exposed to Imaging buffer (100 mM Tris pH7.0, 30 mMNaCl, 0.05% Tween 20, 50 mM sodium ascorbate, freshly dissolved).

Templates were scanned in 4 colors at RT.

Templates were then exposed to sequencing cycles of Cleavage andIncorporation as follows:

Cleavage

-   Prime with Cleavage buffer (0.1M Tris pH 7.4, 0.1 M NaCl and 0.05%    Tween 20). Heat to 60° C.-   Treat the clusters with Cleavage mix (100 mM TCEP in Cleavage    buffer).-   Wait for a total of 15 min in addition to pumping fresh buffer every    4 min.-   Cool to 20° C.-   Wash with Enzymology buffer.-   Wash with 5×SSC/0.05% Tween 20.-   Prime with Imaging buffer.-   Scan in 4 colors at RT.    Incorporation-   Prime with Incorporation buffer Heat to 60° C.-   Treat with Incorporation mix. Wait for a total of 15 min in addition    to pumping fresh Incorporation mix every 4 min.-   Cool to 20° C.-   Wash with Incorporation buffer.-   Wash with 5×SSC/0.05% Tween 20.-   Prime with imaging buffer.-   Scan in 4 colors at RT.-   Repeat the process of Incorporation and Cleavage for as many cycles    as required. Incorporated nucleotides were detected using the    fluorescent imaging apparatus described above.

Alternatively, the flowcell can be sequenced in a fully automated way,with the first incorporation being performed on this instrument, asdescribed below:

After setting the flowcell on the instrument manifold, the templates canbe exposed to the sequencing cycles described below: first baseincorporation, imaging then alternating cleavage, imaging andincorporation, imaging steps for as many sequencing cycles as required.

First Base Incorporation

-   Pump 1000 ul of incorporation buffer at RT-   Set temperature at 55° C. and hold-   Wait for 2 minutes-   Pump 600 ul of incorporation mix-   Wait for 4 minutes-   Pump 200 ul of incorporation mix-   Wait for 4 minutes-   Pump 200 ul of incorporation mix-   Wait for 4 minutes-   Set temperature at 22° C.-   Wait for 2 minutes-   Pump 600 ul of incorporation buffer-   Pump 600 ul of high salt buffer-   Pump 800 ul of scanning mix-   Stop active cooling    Imaging Step

Cleavage

-   Pump 1000 ul of cleavage buffer at RT-   Set temperature at 55° C. and hold-   Wait for 2 minutes-   Pump 600 ul of cleavage mix-   Wait for 4 minutes-   Pump 200 ul of cleavage mix-   Wait for 4 minutes-   Pump 200 ul of cleavage mix-   Wait for 4 minutes-   Set temperature at 22° C. and hold-   Wait for 2 minutes-   Pump 600 ul of incorporation buffer-   Pump 600 ul of high salt buffer-   Pump 800 ul of scanning mix-   Stop active cooling    Imaging Step

Incorporation

-   Pump 1000 ul of incorporation buffer at RT-   Set temperature at 55° C. and hold-   Wait for 2 minutes-   Pump 600 ul of incorporation mix-   Wait for 4 minutes-   Pump 200 ul of incorporation mix-   Wait for 4 minutes-   Pump 200 ul of incorporation mix-   Wait for 4 minutes-   Set temperature at 22° C. and hold-   Wait for 2 minutes-   Pump 600 ul of incorporation buffer-   Pump 600 ul of high salt buffer-   Pump 800 ul of scanning mix-   Stop active cooling.

Each tile of each the chip for the non-fully automated process above wasrecorded in each of the four colors corresponding to the labelednucleotides. The images were analyzed to pick the brightest color foreach cluster, and this image intensity analysis was used to call thebase for each cluster at each cycle. Images from each cycle wereco-localized to obtain the sequence corresponding to each cluster. Asthe sequence of each cluster was known; and was the same for everycluster in the above experiment, the error rates (i.e. clusters notcalled as the correct sequence) could be analyzed for each cycle ofnucleotide incorporation. The error rates were less than 1% for thefirst 20 cycles of the experiment, meaning the known sequence of themonotemplate was correctly called.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

1. A system for detecting fluorescence emitted from nucleic acids,wherein the nucleic acids comprise fluorescent labels, the systemcomprising: a) a stage configured to hold a solid substrate having thenucleic acids; b) a fluid direction system for controllably moving oneor more reagents into contact with the nucleic acids; c) an illuminationsystem for exciting the fluorescent labels via total internal reflection(TIR), the illumination system comprising a mode scrambler and amultimode optical fiber that provides a fluorescent-excitationillumination beam having multiple propagating modes, the mode scramblerscrambling the modes of the illumination beam to provide a substantiallyuniform illumination footprint to excite the fluorescent labels, whereinthe illumination beam is directed to be incident on the solid substrateso that the footprint excites the fluorescent labels through TIR; and d)a detector component for detecting fluorescence produced from excitationof the fluorescent labels by the footprint.
 2. The system of claim 1,further comprising the solid substrate, the solid substrate comprising aflowcell having at least one fluidic channel.
 3. The system of claim 2,wherein the flowcell comprises a channel layer and a cover layer that isoverlaid upon the channel layer, the channel layer including the fluidicchannel, the cover layer having a through-hole that provides access tothe fluidic channel.
 4. The system of claim 2, wherein the fluidicchannel has one or more inlet ports at a first end of the channel andone or more outlet ports at a second end of the channel, the inlet andoutlet ports being located with respect to each other to facilitatemaintaining a uniform flow of a liquid through the fluidic channel. 5.The system of claim 2 wherein the fluid direction system comprises apump placed after the flowcell to pull reagents through the flowcell. 6.The system according to claim 1, further comprising the solid substrate,the solid substrate comprising an array of beads.
 7. The system of claim1, further comprising the nucleic acids and the reagents, the nucleicacids including polynucleotides, the reagents comprising components toextend a second sequence complementary to the one or morepolynucleotides.
 8. The system according to claim 7, wherein saidreagents are fluorescently labeled nucleoside triphosphates.
 9. Thesystem according to claim 7, wherein said reagents are fluorescentlylabeled oligonucleotides.
 10. The system of claim 7, wherein thereagents include four different reagents having respective fluorescentlabels, the detector component further comprising four optic filtersappropriate for filtering emissions of the four fluorescently labeledreagents and light from the illumination beam.
 11. The system of claim1, wherein the illumination system comprises at least one excitationlaser coupled through the multimode fiber.
 12. The system according toclaim 11, further comprising a computer component that is operablycoupled to the mode scrambler, the computer component controlling themode scrambler to physically engage the multimode fiber to change anindex of refraction of the multimode fiber a plurality of times toscramble the modes.
 13. The system according to claim 12, wherein thecomputer component controls the mode scrambler to squeeze or compressthe multimode fiber.
 14. A system according to claim 13, wherein thecomputer component controls the mode scrambler to change an intensity ofthe squeezing or compressing over time to dynamically scramble themodes.
 15. The system according to claim 12, wherein the computercomponent controls the mode scrambler to vibrate the multimode fiber.16. The system of claim 11, wherein said multimode fiber includes a corehaving a substantially rectangular cross-sectional shape.
 17. The systemof claim 1, wherein the detector component comprises a CCD camera. 18.The system of claim 1, wherein said detector component comprises atleast two CCD cameras.
 19. The system of claim 1, wherein the detectorcomponent further comprises one or more optic filters appropriate tofilter fluorescence emissions from the nucleic acids and light from theillumination beam.
 20. The system of claim 1, wherein the fluiddirection system includes two fluidics stations for operating on twoflowcells simultaneously.
 21. The system of claim 1, wherein theillumination system comprises two excitation lasers coupled through afiberoptic device, wherein the excitation lasers illuminate at leastpart of the footprint.
 22. The system of claim 1, further comprising: atemperature control system for regulating the temperature of at leastone of the solid substrate and the reagents, and; a computer componentoperably coupled to the detector component, wherein the computercomponent comprises an instruction set for acquiring fluorescence imagesfrom the detector component; wherein the nucleic acids comprise DNAclusters and the reagents comprise bases having the fluorescent labels,each DNA cluster including a plurality of single-stranded DNA fragmentshaving a common sequence; and wherein the fluid direction system, thetemperature control system, the illumination system, the detectorcomponent, and the computer component cooperate with one another toperform a DNA sequencing cycle including the steps of: (i) incorporatingat least one base onto the DNA fragments of the DNA clusters; (ii)illuminating the DNA clusters to excite the fluorescent labels of thebases; (iii) capturing an image of emitted fluorescence from the DNAclusters; (iii) identifying the base incorporated onto the DNA fragmentsof the DNA clusters based upon the emitted fluorescence from the DNAclusters; and (iv) removing the fluorescent labels from the bases. 23.The system of claim 22, wherein the base is one of four types of bases,the bases of each type having a unique fluorescent label.
 24. The systemof claim 22, wherein the computer component is configured to repeatsteps (i)-(iv) to determine the sequences of the DNA fragments.
 25. Thesystem of claim 22 wherein the detector component is a CCD camera and adensity of DNA clusters recorded on the CCD camera is about 5,000 to50,000 clusters per million pixels.
 26. The system of claim 1, whereinthe reagents include four different bases in which each base has adifferent fluorescent label, the illumination system including first andsecond lasers, each of the first and second lasers exciting two of thefour fluorescent labels so that the four fluorescent labels are excitedby the first and second lasers.
 27. The system of claim 26 furthercomprising four different optical filters for selecting the fluorescenceemission wherein two of the four filters have a narrower bandwidth thanthe other two of the four filters.
 28. A system according to claim 1,further comprising a computer component that is operably coupled to themode scrambler, the computer component configured to control the modescrambler to change an index of refraction along the multimode fiber toscramble the modes and provide the substantially uniform illuminationfootprint.
 29. A system according to claim 28, wherein the computercomponent controls the mode scrambler to dynamically change the index ofrefraction such that the index of refraction is changed by a firstdegree and subsequently changed by a second degree, wherein the firstand second degrees are different amounts.
 30. A system according toclaim 28, wherein the computer component controls the mode scrambler tochange the index of refraction along the multimode fiber a plurality oftimes within a predetermined image capture time period.
 31. A systemaccording to claim 28, wherein the computer component controls the modescrambler to change the index of refraction at a plurality of nodesalong the multimode fiber.
 32. A system according to claim 1, whereinthe mode scrambler includes a waveplate, the illumination beampropagating through the waveplate before illuminating the footprint. 33.A system according to claim 32, wherein the waveplate is rotating whenthe illumination beam propagates therethrough.
 34. A system according toclaim 1, wherein the footprint is sized to excite at least twentythousand clusters of DNA that are separate from each other.
 35. A systemaccording to claim 1, wherein the footprint is sized to be at least 0.25mm².
 36. A system for detecting fluorescence emitted from a sample, thesystem comprising: a) a stage configured to hold a solid substratehaving the sample; b) a fluid direction system for controllably movingone or more reagents into contact with the sample; c) an illuminationsystem for exciting fluorescent labels in the sample via total internalreflection (TIR), the illumination system comprising a mode scramblerand a multimode optical fiber that provides a fluorescent-excitationillumination beam having multiple propagating modes, the mode scramblerscrambling the modes of the illumination beam to provide a substantiallyuniform illumination footprint to excite the fluorescent labels, whereinthe illumination beam is directed to be incident on the solid substrateso that the footprint excites the fluorescent labels through TIR; and d)a detector component for detecting fluorescence produced from excitationof the fluorescent labels by the footprint thereby providing fluorescentdata; e) a computer component comprising an instruction set to processthe fluorescent data and determine sequence information for nucleicacids in the sample.
 37. The system of claim 36, further comprising thesolid substrate, the solid substrate comprising a flowcell having atleast one fluidic channel.
 38. The system of claim 36, furthercomprising the reagents and the sample having the nucleic acids, thenucleic acids including polynucleotides, the reagents comprisingcomponents to extend a second sequence complementary to the one or morepolynucleotides.
 39. The system according to claim 38, wherein saidreagents are fluorescently labeled nucleoside triphosphates.
 40. Thesystem of claim 38, wherein the reagents include four different reagentshaving respective fluorescent labels, the detector component furthercomprising four optic filters appropriate for filtering emissions of thefour fluorescently labeled reagents and light from the illuminationbeam.
 41. The system of claim 36, wherein the illumination systemcomprises at least one excitation laser coupled through the multimodefiber.
 42. The system according to claim 41, wherein the computercomponent is operably coupled to the mode scrambler, the computercomponent controlling the mode scrambler to physically engage themultimode fiber to change an index of refraction of the multimode fibera plurality of times to scramble the modes.
 43. The system of claim 41,wherein said multimode fiber includes a core having a substantiallyrectangular cross-sectional shape.
 44. The system of claim 36, whereinsaid detector component comprises at least two CCD cameras.
 45. Thesystem of claim 36, wherein the fluid direction system includes twofluidics stations for operating on two flowcells simultaneously.
 46. Thesystem of claim 36, wherein the illumination system comprises twoexcitation lasers coupled through a fiberoptic device, wherein theexcitation lasers illuminate at least part of the footprint.
 47. Thesystem of claim 36, further comprising: a temperature control system forregulating the temperature of at least one of the substrate and thereagents; wherein the computer component is operably coupled to thedetector component, wherein the computer component comprises aninstruction set for acquiring fluorescence images from the detectorcomponent; wherein the nucleic acids comprise DNA clusters and thereagents comprise bases having the fluorescent labels, each DNA clusterincluding a plurality of single-stranded DNA fragments having a commonsequence; and wherein the fluid direction system, the temperaturecontrol system, the illumination system, the detector component, and thecomputer component cooperate with one another to perform a DNAsequencing cycle including the steps of: (i) incorporating at least onebase onto the DNA fragments of the DNA clusters; (ii) illuminating theDNA clusters to excite the fluorescent labels of the bases; (iii)capturing an image of emitted fluorescence from the DNA clusters; (iii)identifying the base incorporated onto the DNA fragments of the DNAclusters based upon the emitted fluorescence from the DNA clusters; and(iv) removing the fluorescent labels from the bases.
 48. A systemaccording to claim 36, wherein the computer component is operablycoupled to the mode scrambler, the computer component configured tocontrol the mode scrambler to change an index of refraction along themultimode fiber to scramble the modes and provide the substantiallyuniform illumination footprint.
 49. A system according to claim 48,wherein the computer component controls the mode scrambler todynamically change the index of refraction such that the index ofrefraction is changed by a first degree and subsequently changed by asecond degree, wherein the first and second degrees are differentamounts.
 50. A system according to claim 48, wherein the computercomponent controls the mode scrambler to change the index of refractionalong the multimode fiber a plurality of times within a predeterminedimage capture time period.
 51. A system according to claim 48, whereinthe computer component controls the mode scrambler to change the indexof refraction at a plurality of nodes along the multimode fiber.
 52. Asystem according to claim 36, wherein the mode scrambler includes awaveplate, the illumination beam propagating through the waveplatebefore illuminating the footprint.
 53. A system according to claim 36,wherein the footprint is sized to excite at least twenty thousandclusters of DNA that are separate from each other.
 54. A systemaccording to claim 36, wherein the footprint is sized to be at least0.25 mm².